Cd20 binding molecules and uses thereof

ABSTRACT

This disclosure provides pentameric and hexameric CD20 binding molecules and methods of using such molecules to direct complement-mediated, T-cell-mediated, or both complement-mediated and T-cell-mediated killing of CD20-expressing cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/554,301, filed Aug. 29, 2017, which is a US National Stage Entry ofPCT Application No. PCT/US2016/020920, filed Mar. 4, 2016, which claimsbenefit to U.S. Provisional Appl. No. 62/128,284, filed on Mar. 4, 2015;the disclosures of which applications are incorporated herein byreference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 001US2-Sequence-Listing; Size: 95,923 bytes; and Dateof Creation: Jul. 22, 2020) filed with the application is incorporatedherein by reference in its entirety.

BACKGROUND

Since the advent of humanized antibodies, the therapeutic use ofantibodies such as RITUXAN® (rituximab) has revolutionized the treatmentof B-cell malignancies. Rituximab, a CD20 specific chimeric monoclonalantibody, is the first effective targeted therapy approved by the FDAfor treatment of relapsed or refractory B-cell non-Hodgkin's lymphoma.This scientific achievement has not only changed the standard ofpractice for treatment of B-cell lymphoma but has stimulated significantinterest in next generation of CD20 mAbs. Many novel CD20 mAbs haveentered the clinic, each with structural modifications to furtherimprove efficacy. In this application, we describe a series of CD20antibodies that can exhibit improved efficacy utilizing additional modesof action, as these CD20 antibodies are expressed in non-IgG formats,such as IgA and IgM.

CD20 is a 36 kDa non-glycosylated; tetra-spanning membrane protein(MS4A1 gene product) expressed exclusively on B-lymphocytes and morethan 90% of B-lymphocytic lymphoma (see FIG. 1). CD20 is expressed atthe late pre-B cell stage and is upregulated on most normal andmalignant B lineage cells before it is down-regulated in terminallydifferentiated plasma cells. This B-lymphocyte surface molecule isinvolved with development and differentiation of B-cells into plasmacells.

Although rituximab exhibits significant anti-tumor activity in patientswith B-cell non-Hodgkin's lymphoma, there is a need to improve theefficacy of antibody therapeutics for the treatment of B-cell neoplasms.Rituximab as single agent therapy results in a clinical response rate of50%, and it is unclear why the remaining 50% of patients do not respond.In addition, a majority of responsive patients acquires resistance tofurther rituximab therapy. One mechanism of initial or acquiredresistance is the down regulation or modulation of CD20 on the tumorcells (or clonal expansion of low expressing tumors). There is a clearunmet medical need for improved treatment for these refractory patientswhose refractory tumors have minimizes CD20 expression. By increasingaffinity and avidity with the multivalency of IgA or IgM, these neweragents aim to achieve improved response rates as compared to rituximab.Furthermore, altering the effector functions by changing the antibodyisotype may improve the potency and efficacy of CD20 antibodies.

Since the introduction of rituximab, much has been learned aboutpotential mechanisms for the therapeutic efficacy of CD20 mAbs.Rituximab induces B-cell death primarily through complement-dependentlysis (CDC) and antibody dependent cellular toxicity (ADCC) effectormechanisms, and to a lesser degree via cellular apoptosis. CD20antibodies are described as either Type I (such as rituximab/RITUXAN® orofatumumab/ARZERRA®), which redistributes CD20 into detergent-resistantlipid rafts; and Type II such as tositumumab (B1) andobinutuzumab/GAZYVA®, which do not. Clustering by Type I antibodiespromotes association with other cell surface proteins such as the B-cellreceptor (BCR), and binding to C1q, resulting in potentcomplement-dependent cytotoxicity (CDC). IgM is highly potent atinducing complement dependent cytotoxicity, and as such IgM forms ofCD20 antibodies can yield significantly greater efficacy. In contrast,Type II antibodies, such as tositumomab (B1), elicit antibody-dependentcellular cytotoxicity, but not complement-dependent cytotoxicity. TypeII antibodies are very potent at directly triggering cell death viaantibody-induced homotypic adhesion and lysosomal cell death. Oligomericforms of antibodies, such as IgA and IgM exhibit increased multivalencyand can display enhanced efficacy at inducing homotypic adhesion andcell death. Importantly, both type I and type II antibodies recruitFcR-expressing cells to mediate cellular effector functions such asantibody-dependent cellular cytotoxicity and antibody-dependent cellularphagocytosis.

Second-generation type I CD20 mAbs have been approved for human use.Ofatumumab is a fully human IgG1, type I CD20 mAb that exhibits antibodydependent cellular cytotoxicity (ADCC) but has strongercomplement-dependent cytotoxicity (CDC) when compared to rituximab. As atype I antibody, ofatumumab is relatively ineffective in triggering celldeath. Ofatumumab is FDA approved for treatment of chronic lymphocyticleukemia (CLL). Obinutuzumab/GA101 is a humanized IgG1 second-generationtype II mAb which displays improved pro-apoptotic activity, enhancedADCC but no complement-fixing activity. Obinutuzumab is FDA approved fortreatment CLL. Another fully human IgG1κ CD20 antibody huMAb 1.5.3 (seeU.S. Patent Publication No. 2007-0014720), was designed for enhancepotency. Preclinical studies have shown that huMAb 1.5.3 has greaterapoptosis as compared to rituximab and exhibits both potent CDC as wellas ADCC activity. HuMAb 1.5.3 appears to combine both type I and type IIactivities including effective cell killing through direct apoptosisinduction, CDC and ADCC. IgM and IgA can contribute to further enhancedpotency of CD20 antibodies with increased avidity which can mediateincreased sensitivity and cytotoxicity on low CD20 expressing tumorcells. Furthermore, the efficacy of type I and type II CD20 activitiescan be increased with IgA or IgM formats allowing the development ofmost potent anti-tumor antibodies utilizing optimally combinedmechanisms of action, including the triggering direct apoptosis,enhanced CDC and ADCC.

SUMMARY

This disclosure provides a multimeric binding molecule that includes atleast two bivalent binding units or variants or fragments thereof, whereeach binding unit includes at least two heavy chain constant regions orfragments thereof, each associated with an antigen-binding domain. Atleast one antigen binding domain of the provided binding molecule is aCD20 antigen binding domain that includes six immunoglobulincomplementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3, where the HCDR1 includes the amino acid sequence of SEQ IDNO: 39 or SEQ ID NO: 39 with one or two single amino acid substitutions;the HCDR2 includes the amino acid sequence of SEQ ID NO: 40 or SEQ IDNO: 40 with one or two single amino acid substitutions; the HCDR3includes the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 41 withone or two single amino acid substitutions; the LCDR1 includes the aminoacid sequence of SEQ ID NO: 43, or SEQ ID NO: 43 with one or two singleamino acid substitutions; the LCDR2 includes the amino acid sequence ofSEQ ID NO: 44 or SEQ ID NO: 44 with one or two single amino acidsubstitutions; and the LCDR3 includes the amino acid sequence of SEQ IDNO: 45 or SEQ ID NO: 45 with one or two single amino acid substitutions.

The disclosure further provides a multimeric binding molecule thatincludes at least two bivalent binding units or variants or fragmentsthereof, where each binding unit includes at least two heavy chainconstant regions or fragments thereof, each associated with anantigen-binding domain. At least one antigen binding domain of theprovided binding molecule is a CD20 antigen binding domain that includesan antibody heavy chain variable region (VH) and an antibody light chainvariable region (VL), where the VH includes an amino acid sequence atleast 80%, at least 85%, at least 90%, at least 95% or 100% identical toSEQ ID NO: 38, and the VL includes an amino acid sequence at least 80%,at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO:42.

In certain aspects a binding molecule provided by the disclosure is adimeric binding molecule that includes two bivalent IgA binding units orfragments thereof and a J-chain or fragment or variant thereof, whereeach binding unit includes two IgA heavy chain constant regions orfragments thereof each associated with an antigen-binding domain. Adimeric IgA binding molecule as provided herein can further include asecretory component, or fragment or variant thereof. The IgA heavy chainconstant regions or fragments thereof can each include a Cα2 domain or aCα3-tp domain and can, in certain aspects, further include a Cα1 domain.In certain aspects, the IgA heavy chain constant regions can be humanIgA constant regions. In certain aspects, each binding unit includes twoIgA heavy chains each including a VH situated amino terminal to the IgAconstant region or fragment thereof, and two immunoglobulin light chainseach including a VL situated amino terminal to an immunoglobulin lightchain constant region.

In certain aspects a binding molecule provided by the disclosure is a isa pentameric or a hexameric binding molecule including five or sixbivalent IgM binding units, respectively, where each binding unitincludes two IgM heavy chain constant regions or fragments thereof eachassociated with an antigen-binding domain. The IgM heavy chain constantregions or fragments thereof can each include a Cμ3 domain and a Cμ4-tpdomain and can, in certain aspects further include a Cμ2 domain, a Cμ1domain, or any combination thereof.

Where the binding molecule is pentameric, the binding molecule canfurther include a J-chain, or fragment thereof, or functional fragmentthereof, or a functional variant thereof. In certain aspects, theJ-chain or fragment thereof includes the amino acid sequence SEQ ID NO:49 or a functional fragment thereof. In certain aspects, the J-chain orfragment thereof can further include a heterologous polypeptide. Theheterologous polypeptide can be directly or indirectly fused to theJ-chain or fragment thereof. In certain aspects the heterologouspolypeptide can be indirectly fused to the J-chain or fragment thereofvia a peptide linker. In certain aspects the peptide linker can include,e.g., at least 5 amino acids, but no more than 25 amino acids. Incertain aspects the peptide linker consists of GGGGSGGGGSGGGGS (SEQ IDNO: 67). The heterologous polypeptide can be fused to or near theN-terminus of the J-chain or fragment thereof, the C-terminus of theJ-chain or fragment thereof, or to both the N-terminus and C-terminus ofthe J-chain or fragment thereof. In certain aspects the heterologouspolypeptide can include a binding domain, e.g., an antibody orantigen-binding fragment thereof. The antigen-binding fragment can be,for example, a Fab fragment, a Fab′ fragment, an F(ab′)₂ fragment, an Fdfragment, an Fv fragment, a single-chain Fv (scFv) fragment, adisulfide-linked Fv (sdFv) fragment, or any combination thereof. Incertain aspects the heterologous polypeptide can specifically bind toCD3ε. For example, in certain aspects the modified J-chain can includethe amino acid sequence SEQ ID NO: 64 (V15J) or SEQ ID NO: 66 (J15V).Moreover, in certain aspects, these particular modified J-chains canfurther include a signal peptide, where the modified J-chain thenincludes the amino acid sequence SEQ ID NO: 63 (V15J) or SEQ ID NO: 65(J15V).

In certain aspects, the IgM heavy chain constant regions can be humanIgM heavy chain constant regions. In certain aspects, each binding unitincludes two IgM heavy chains each including a VH situated aminoterminal to the IgM constant region or fragment thereof, and twoimmunoglobulin light chains each including a VL situated amino terminalto an immunoglobulin light chain constant region.

In certain aspects, at least one binding unit of a multimeric bindingmolecule provided herein includes two of the CD20 antigen bindingdomains, which can be the same or different. In certain aspects, atleast three, at least four, at least five, at least six, at least seven,at least eight, at least nine, at least ten, at least eleven, or atleast twelve copies of the same CD20 antigen binding domain.

In certain aspects where the binding molecule is an IgM bindingmolecule, the two IgM heavy chains within at least one binding unitinclude the amino acid sequence SEQ ID NO: 56.

In certain aspects where the binding molecule includes immunoglobulinlight chains, the two light chain constant regions of a given bindingunit can be human lambda constant regions or a human kappa constantregion. In certain aspects the two light chain constant regions areidentical and include the amino acid sequence SEQ ID NO: 58.

In certain aspects, at least two, at least three, at least four, atleast five, or at least six of the binding units of a multimeric bindingmolecule provided herein are identical.

In certain aspects, the binding molecule as described above can directcomplement-mediated, T-cell-mediated, or both complement-mediated andT-cell-mediated killing of a CD-20-expressing cell at higher potencythan an equivalent amount of a monospecific, bivalent IgG1 antibody orfragment thereof that specifically binds to the same CD20 epitope as theone or more CD20 antigen binding domains of the binding molecule. Incertain aspects, the monospecific, bivalent IgG1 antibody is 1.5.3,which includes a VH having the amino acid sequence SEQ ID NO: 38 and aVL having the amino acid sequence SEQ ID NO: 42. In certain aspects, theCD-20-expressing cell is a lymphoma cell line, for example, a Ramos cellline, a Raji cell line, a Daudi cell line, a Namalwa cell line, a Grantacell line, a Z138 cell line, a DoHH2 cell line, or a DB cell line. Incertain aspects, where the CD20-expressing cell is a Raji cell line, thebinding molecule can direct complement-mediated killing with an IC₅₀ atleast four-fold, at least ten-fold, at least 50-fold, or at least100-fold lower than the IC₅₀ of an equivalent amount of the monospecificbivalent IgG1 antibody, as measured, e.g., in μg/ml. In certain aspects,where the CD20-expressing cell is a Ramos cell line, the bindingmolecule can direct complement-mediated killing with an IC₅₀ at leastten-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least90-fold, or at least 100-fold lower than the IC₅₀ of an equivalentamount of the monospecific bivalent IgG1 antibody, as measured, e.g., asmolar equivalents. In certain aspects, the CD-20-expressing cell is amalignant B cell in a subject with cancer, e.g., a CD20-positiveleukemia, lymphoma, or myeloma. In certain aspects, the cancer isminimally responsive or non-responsive to rituximab therapy. In certainaspects, the subject is human.

The disclosure further provides a composition that includes the bindingmolecule as described above.

The disclosure further provides a polynucleotide including a nucleicacid sequence that encodes a polypeptide subunit of a multimeric bindingmolecule as provided herein. In certain aspects, the disclosure providesa polynucleotide that includes a nucleic acid sequence encoding a heavychain polypeptide subunit of a multimeric binding molecule as providedherein, where the heavy chain polypeptide subunit includes an IgM heavychain constant region or fragment thereof or an IgA heavy chain constantregion or fragment thereof, and at least the antibody VH portion of theCD20 antigen binding domain. In certain aspects, the heavy chainpolypeptide subunit can include a human IgM constant region or fragmentthereof fused to the C-terminal end of a VH that includes (a) an HCDR1,HCDR2, HCDR3, where the HCDR1 includes the amino acid sequence of SEQ IDNO: 39 or SEQ ID NO: 39 with one or two single amino acid substitutions;the HCDR2 includes the amino acid sequence of SEQ ID NO: 40 or SEQ IDNO: 40 with one or two single amino acid substitutions; the HCDR3includes the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 41 withone or two single amino acid substitutions; or (b) an amino acidsequence at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to SEQ ID NO: 38. In certain aspects the providedpolynucleotide can encode the amino acid sequence SEQ ID NO: 56. Thedisclosure further provides a polynucleotide composition that includes apolynucleotide as described.

The provided polynucleotide composition can further include, e.g., anucleic acid sequence that encodes a light chain polypeptide subunit,where the light chain polypeptide subunit includes the antibody VLportion of the CD20 antigen binding domain. In certain aspects the lightchain polypeptide subunit can include a human antibody light chainconstant region or fragment thereof fused to the C-terminal end of a VLthat includes: (a) an LCDR1, LCDR2, and LCDR3, where the LCDR1 includesthe amino acid sequence of SEQ ID NO: 43, or SEQ ID NO: 43 with one ortwo single amino acid substitutions; the LCDR2 includes the amino acidsequence of SEQ ID NO: 44 or SEQ ID NO: 44 with one or two single aminoacid substitutions; and the LCDR3 includes the amino acid sequence ofSEQ ID NO: 45 or SEQ ID NO: 45 with one or two single amino acidsubstitutions; or (b) an amino acid sequence at least 80%, at least 85%,at least 90%, at least 95% or 100% identical to SEQ ID NO: 42. Incertain aspects the nucleic acid sequence that encodes the light chainpolypeptide subunit can encode the amino acid sequence SEQ ID NO: 58.

In certain aspects of the provided polynucleotide composition, thenucleic acid sequence encoding the heavy chain polypeptide subunit andthe nucleic acid sequence encoding the light chain polypeptide subunitcan be on separate vectors, or they can be situated on a single vector.

The provided polynucleotide composition can further include, e.g., anucleic acid sequence that encodes a J-chain, or functional fragmentthereof, or a functional variant thereof. In certain aspects the J-chainor fragment thereof can include the amino acid sequence SEQ ID NO: 49 ora functional fragment thereof. Moreover, the J-chain or fragment thereofcan be a modified J-chain that further includes a heterologouspolypeptide. The heterologous polypeptide can, in certain aspects, bedirectly or indirectly fused to the J-chain or fragment thereof. Incertain aspects, the heterologous polypeptide can include an antibody orantigen-binding fragment thereof. In certain aspects, the heterologouspolypeptide can be, e.g., a scFv that can specifically bind to CD3ε. Incertain aspects the modified J-chain can include the amino acid sequenceSEQ ID NO: 64 (V15J) or SEQ ID NO: 66 (J15V). In those aspects where themodified J-chain further comprises a signal peptide, the modifiedJ-chain can include the amino acid sequence SEQ ID NO: 63 (V15J) or SEQID NO: 65 (J15V). In certain aspects of the provided polynucleotidecomposition, the nucleic acid sequence that encodes a J-chain, orfunctional fragment thereof, or a functional variant thereof can includeSEQ ID NO: 68 or SEQ ID NO: 69.

In certain aspects of the provided polynucleotide composition, thenucleic acid sequence encoding the heavy chain polypeptide subunit, thenucleic acid sequence encoding the light chain polypeptide subunit, andthe nucleic acid sequence encoding the J-chain can be situated on asingle vector, or they can be situated on two or three separate vectors.The disclosure further provides the vector or vectors that singly orcollectively contain the provided polynucleotide composition. Thedisclosure further provides a host cell including the providedpolynucleotide, the provided polynucleotide composition, or the providedvector or vectors, where the host cell can express a multivalentanti-CD20 binding molecule as provided herein. The disclosure furtherprovides a method of producing a multivalent anti-CD20 binding moleculeas provided herein, where the method includes culturing the providedhost cell, and recovering the binding molecule.

In certain aspects, the disclosure provides a method for directingcomplement-mediated, T-cell-mediated, or both complement-mediated andT-cell-mediated killing of a CD20-expressing cell where the methodincludes contacting a CD20-expressing cell with a multimeric bindingmolecule as provided herein or a composition that includes the providedbinding molecule. According to these aspects, the binding molecule candirect complement-mediated, T-cell-mediated, or both complement-mediatedand T-cell-mediated killing of a CD-20-expressing cell at higher potencythan an equivalent amount of a monospecific, bivalent IgG1 antibody orfragment thereof that specifically binds to the same CD20 epitope as theCD20 antigen binding domain. In certain aspects, the monospecific,bivalent IgG1 antibody is 1.5.3, which includes a VH having the aminoacid sequence SEQ ID NO: 38 and a VL having the amino acid sequence SEQID NO: 42. In certain aspects, the CD-20-expressing cell is a lymphomacell line, e.g., a Ramos cell line, a Raji cell line, a Daudi cell line,a Namalwa cell line, a Granta cell line, a Z138 cell line, a DoHH2 cellline, or a DB cell line. In certain aspects, the CD20-expressing cell isa Raji cell line, and the binding molecule directs complement-mediatedkilling with an IC₅₀ at least four-fold, at least ten-fold, at least50-fold, or at least 100-fold lower than the IC₅₀ of an equivalentamount of the monospecific bivalent IgG1 antibody, as measured, e.g., inμg/ml. In certain aspects, where the CD20-expressing cell is a Ramoscell line, the binding molecule can direct complement-mediated killingwith an IC₅₀ at least ten-fold, at least 20-fold, at least 30-fold, atleast 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, atleast 80-fold, at least 90-fold, or at least 100-fold lower than theIC₅₀ of an equivalent amount of the monospecific bivalent IgG1 antibody,as measured, e.g., as molar equivalents. In certain aspects, theCD-20-expressing cell is a malignant B cell in a subject with cancer,e.g., a CD20-positive leukemia, lymphoma, or myeloma. In certainaspects, the cancer is minimally responsive or non-responsive torituximab therapy. In certain aspects the subject is a human.

In other aspects, the disclosure provides a method for directingcomplement-mediated, T-cell-mediated, or both complement-mediated andT-cell-mediated killing of a CD20-expressing cell, where the methodincludes contacting a CD20-expressing cell with a dimeric, pentameric,or hexameric binding molecule including two, five, or six bivalentbinding units, respectively, where each binding unit includes two IgA orIgM heavy chain constant regions or fragments thereof and two antigenbinding domains, where at least one antigen binding domain of thebinding molecule is a CD20 antigen binding domain, and where the bindingmolecule can direct complement-mediated, T-cell-mediated, or bothcomplement-mediated and T-cell-mediated killing of a CD-20-expressingcell at higher potency than an equivalent amount of a monospecific,bivalent IgG1 antibody or fragment thereof that specifically binds tothe same CD20 epitope as the CD20 antigen binding domain.

According to these provided methods, the CD20 antigen binding domain caninclude six immunoglobulin complementarity determining regions HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where the HCDR1 includes theamino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 2 with one or twosingle amino acid substitutions; the HCDR2 includes the amino acidsequence of SEQ ID NO: 3 or SEQ ID NO: 3 with one, two, three, four, orfive single amino acid substitutions; the HCDR3 includes the amino acidsequence of SEQ ID NO: 4, SEQ ID NO: 4 with one, two, or three singleamino acid substitutions, SEQ ID NO: 10, or SEQ ID NO: 31, the LCDR1includes the amino acid sequence of SEQ ID NO: 6, or SEQ ID NO: 6 withone, two, or three single amino acid substitutions; the LCDR2 includesthe amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7 with one or twosingle amino acid substitutions; and the LCDR3 includes the amino acidsequence of SEQ ID NO: 8 or SEQ ID NO: 8 with one or two single aminoacid substitutions.

According to these provided methods, the CD20 antigen binding domain caninclude a VH and a VL including, respectively: (a) an amino acidsequence at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to SEQ ID NO: 1 and an amino acid sequence at least 80%, atleast 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 5;(b) an amino acid sequence at least 80%, at least 85%, at least 90%, atleast 95% or 100% identical to SEQ ID NO: 9 and an amino acid sequenceat least 80%, at least 85%, at least 90%, at least 95% or 100% identicalto SEQ ID NO: 11; (c) an amino acid sequence at least 80%, at least 85%,at least 90%, at least 95% or 100% identical to SEQ ID NO: 15 and anamino acid sequence at least 80%, at least 85%, at least 90%, at least95% or 100% identical to SEQ ID NO: 18; (d) an amino acid sequence atleast 80%, at least 85%, at least 90%, at least 95% or 100% identical toSEQ ID NO: 22 or SEQ ID NO: 23 and an amino acid sequence at least 80%,at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 29; (e) an amino acidsequence at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to SEQ ID NO: 30 and an amino acid sequence at least 80%, atleast 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO:32; or (f) an amino acid sequence at least 80%, at least 85%, at least90%, at least 95% or 100% identical to SEQ ID NO: 35 and an amino acidsequence at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to SEQ ID NO: 37.

According to these provided methods, the IgA heavy chain constantregions or fragments thereof of the binding molecule can each include aCα2 domain or a Cα3-tp domain, and one or more IgA heavy chain constantregions or fragments thereof can further include a Cα1 domain. Incertain aspects, the IgA heavy chain constant region is a human IgAconstant region. In certain aspects a dimeric binding molecule accordingto these provided methods can further include a secretory component

According to these provided methods, the IgM heavy chain constantregions or fragments thereof of the binding molecule each include a Cμ3domain and a Cμ4-tp domain, and in certain aspects can further include aCμ2 domain, a Cμ1 domain, or any combination thereof. In certain aspectsthe IgM heavy chain constant region is a human IgM constant region.

According to these provided methods, where the binding molecule isdimeric or pentameric, the binding molecule can further include aJ-chain, or functional fragment thereof, or a functional variantthereof. In certain aspects, the J-chain or fragment thereof includesthe amino acid sequence SEQ ID NO: 49 or a functional fragment thereof.In certain aspects, the J-chain or fragment thereof can further includea heterologous polypeptide. The heterologous polypeptide can be directlyor indirectly fused to the J-chain or fragment thereof. In certainaspects the heterologous polypeptide can be indirectly fused to theJ-chain or fragment thereof via a peptide linker. In certain aspects thepeptide linker can include, e.g., at least 5 amino acids, but no morethan 25 amino acids. In certain aspects the peptide linker consists ofGGGGSGGGGSGGGGS (SEQ ID NO: 67). The heterologous polypeptide can befused to or near the N-terminus of the J-chain or fragment thereof, theC-terminus of the J-chain or fragment thereof, or to both the N-terminusand C-terminus of the J-chain or fragment thereof. In certain aspectsthe heterologous polypeptide can include a binding domain, e.g., anantibody or antigen-binding fragment thereof. The antigen-bindingfragment can be, for example, a Fab fragment, a Fab′ fragment, anF(ab′)₂ fragment, an Fd fragment, an Fv fragment, a single-chain Fv(scFv) fragment, a disulfide-linked Fv (sdFv) fragment, or anycombination thereof. In certain aspects the heterologous polypeptide canspecifically bind to CD3ε. For example, in certain aspects the modifiedJ-chain can include the amino acid sequence SEQ ID NO: 64 (V15J) or SEQID NO: 66 (J15V). Moreover, in certain aspects, these particularmodified J-chains can further include a signal peptide, where themodified J-chain then includes the amino acid sequence SEQ ID NO: 63(V15J) or SEQ ID NO: 65 (J15V).

According to these provided methods, each binding unit of the bindingmolecule can include two IgA or IgM heavy chains each including a VHsituated amino terminal to the IgA or IgM constant region or fragmentthereof, and two immunoglobulin light chains each including a VLsituated amino terminal to an immunoglobulin light chain constantregion. In certain aspects, at least one binding unit of the bindingmolecule includes two identical CD20 antigen binding domains, and wherethe two IgA or IgM heavy chains within the at least one binding unit areidentical. In some aspects, the two IgM heavy chains within at least onebinding unit include the amino acid sequence SEQ ID NO: 52. In someaspects, the two light chain constant regions are human lambda constantregions or human kappa constant regions that can be identical andinclude the amino acid sequence SEQ ID NO: 54.

According to these provided methods, the binding molecule can include atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least ten, at leasteleven, or at least twelve CD20 antigen binding domains that in certainaspects can be identical. According to these provided methods at leasttwo, at least three, at least four, at least five, or at least six ofthe binding units within the binding molecule can be identical.

According to these provided methods, the reference monospecific,bivalent IgG1 antibody can be rituximab, which includes a VH having theamino acid sequence SEQ ID NO: 1 and a VL having the amino acid sequenceSEQ ID NO: 5.

According to these provided methods, the CD-20-expressing cell can be alymphoma cell line, e.g., a Ramos cell line, a Raji cell line, a Daudicell line, a Namalwa cell line, a Granta cell line, a Z138 cell line, aDoHH2 cell line, or a DB cell line. In certain aspects where the cellline is a Granta cell line, the binding molecule can directcomplement-mediated killing of the cell line at about six times thepotency of rituximab. In certain aspects where the cell line is a Rajicell line or a Ramos cell line, and where the binding molecule candirect complement-mediated killing of the cell line at about three timesthe potency of rituximab.

According to these provided methods, the CD-20-expressing cell can be amalignant B cell in a subject with cancer, e.g., a CD20-positiveleukemia, lymphoma, or myeloma. In certain aspects, the cancer isminimally responsive or non-responsive to rituximab therapy. In certainaspects, the subject is human.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: The molecular diagram of CD20 molecule. CD20 is atetraspanning-transmembrane protein that predominantly remains on themembrane of B cells without internalization upon antibody binding. Thebinding sites of CD20 monoclonal antibodies, rituximab and ofatumumab,are indicated. A linear diagram of CD20 is included to display theorientation of transmembrane (TM), extracellular domains (ECD) andcytoplasmic regions.

FIG. 2: Schematic diagrams of IgG, IgM hexamer and IgM pentamer. IgG isdisplayed as 150 kDa protein with heavy and light chains indicated. IgMincluding a J-chain is indicated as a pentamer of approximately 915 kDa.IgM without a J-chain is shown as a hexamer having a molecular weight ofapproximately 1080 kDa.

FIG. 3: Non-reducing SDS-PAGE of IgG and IgM. IgG and IgM CD20antibodies based on rituximab run on non-reducing, SDS denaturedpolyacrylamide gel electrophoresis. The first and last lanes aremolecular weight standards. The murine and human IgG1 antibodies (secondand third lanes, respectively) exhibit molecular weights ofapproximately 150 kDa. The IgM+J-chain version (a commercially availableCD20 IgM antibody available from Invivogen) exhibits a molecular weightof about 1050 kDa (fourth lane). The anti-CDIM antibody IGM-55.5 (seePCT Publication No. WO 2013/120012) (fifth lane) is included as anotherIgM for comparison.

FIG. 4: Assembly of Anti-CD20 IgM Oligomers. Non-reducing SDS-PAGE showsthat anti-CD20 antibodies comprising the variable domains of rituximaband 1.5.3 can assemble as IgM. The rituximab IgM is shown in the firstpanel without J-chain (lane 2), and with J-chain (lane 3). The 1.5.3 IgMis shown in the second panel without J-chain (lane 2) and with J-chain(lane 3).

FIG. 5A: ELISA results showing binding of 1.5.3 IgM and 1.5.3 IgG toCD20 at high antigen density (10 μg/ml).

FIG. 5B: ELISA results showing binding of 1.5.3 IgM and 1.5.3 IgG toCD20 at low antigen density (0.3 μg/ml).

FIG. 6A-E Anti-CD20 IgM is more potent than anti-CD20 IgG at complementdependent cytotoxicity. Human cultured leukemia or lymphoma cells wereincubated with commercially-available anti-CD20 IgM or IgG plus 10%human complement. Cell viability was measured after 4 hours using ametabolic indicator dye (CCK8). In most of the lymphoma cell lines(Granta (FIG. 6A), Raji (FIG. 6B), and Ramos (FIG. 6C)), the IgM isotypeof anti-CD20 was more potent than the corresponding IgG isotype, in thepresence of complement. For comparison, the assay was also carried outin Namalwa cells (FIG. 6E), which exhibit minimal expression of CD20,and Nalm-6 cells (FIG. 6D), which are devoid of CD20 expression.

FIG. 7: Anti-CD20 IgM is more potent than rituximab at complementdependent cytotoxicity in the Raji cell line as measured on a μg/mlbasis.

FIG. 8A-B: Anti-CD20 IgM is more effective than rituximab and 1.5.3 IgGat complement dependent cytotoxicity. Ramos cells were incubated withincreasing concentrations of anti-CD20 IgM or IgG and 10% humancomplement. Cell viability was measured after 4 hours. FIG. 8A comparesrituximab (IgG) and a rituximab-like IgM+J-chain.

FIG. 8B compares 1.5.3 (IgG), 1.5.3 IgM, and 1.5.3 IgM+J-chain. Thetables in each panel show EC50 results in molar concentrations.

FIG. 9: Complement-dependent cytotoxicity (CDC) activity of rituximabIgG1 (open circles), rituximab-derived anti-human CD20 IgM+J (closedcircles), 1.5.3 IgG1 (open squares), and 1.5.3 anti-CD20 IgM+J (closedsquares) on DOHH2 and Z138 cells.

FIG. 10: Characterization of 1.5.3 IgM antibodies by gel electrophoresisand western blotting. Lane Key: 1: Marker; 2: 1.5.3 IgM+V15J; 3: 1.5.3IgM+wtJ; 4: 1.5.3 IgM (hexamer); 5: Marker. FIG. 10A shows the hexamerand pentamer forms on a hybrid gel. FIG. 10B shows the antibodiesresolved by non-reducing SDS-PAGE. FIG. 10C shows the antibodiesresolved by reducing SDS-PAGE. The gel in FIG. 10C was transferred to amembrane and J-chain was detected by western blotting, shown in FIG.10D.

FIG. 11: 1.5.3 IgM+V15J (closed squares) elicits T-cell activation in acoculture of CD20+RPMI8226 cells and engineered Jurkat T-cells to agreater extent than blinatumomab (closed circles) or 1.5.3 IgM+wt J(open squares).

FIG. 12: T-cell activation by 1.5.3 IgM V15J as a function of CD20expression using a series of tumor cell lines each expressing adifferent level of CD20 antigen (expressed as mean fluorescenceintensity or MFI).

FIG. 13: Tumor cell killing in human blood using the KILR™ detectionassay. Key: diamonds: 1.5.3 IgM+V15J; squares: 1.5.3 IgM+wild type J;closed circles: 1.5.3 IgG; open circles Rituxan IgG; triangles:blinatumomab.

FIG. 14A-B: T-cell directed B-cell killing in vivo in NSG mice engraftedwith CD34+ cells to generate a human hematopoietic system. The mice weredosed with monospecific or bispecific 1.5.3 IgM and the number of humanB-cells measured before and at 6 hours post dosing. FIG. 14A shows theresults for the monospecific antibody with wild-type J-chain, and FIG.14B shows the results for the bispecific with the V15J anti-CD3 J-chain.

FIG. 15: Comparison of B-cell killing and recovery (at 10-days postdose) between 1.5.3+V15J (top row) and rituximab (bottom row).

DETAILED DESCRIPTION Definitions

The term “a” or “an” entity refers to one or more of that entity; forexample, “a binding molecule,” is understood to represent one or morebinding molecules. As such, the terms “a” (or “an”), “one or more,” and“at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary of Biochemistry andMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation, andderivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a biological source or produced byrecombinant technology but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

A polypeptide as disclosed herein can be of a size of about 3 or more, 5or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides can have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid, e.g., a serine or an asparagine.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated as disclosed herein, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

As used herein, the term “a non-naturally occurring polypeptide” or anygrammatical variants thereof, is a conditional definition thatexplicitly excludes, but only excludes, those forms of the polypeptidethat are, or could be, determined or interpreted by a judge or anadministrative or judicial body, to be “naturally-occurring.”

Other polypeptides disclosed herein are fragments, derivatives, analogs,or variants of the foregoing polypeptides, and any combination thereof.The terms “fragment,” “variant,” “derivative” and “analog” as disclosedherein include any polypeptides which retain at least some of theproperties of the corresponding native antibody or polypeptide, forexample, specifically binding to an antigen. Fragments of polypeptidesinclude, for example, proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of, e.g., a polypeptide include fragments asdescribed above, and also polypeptides with altered amino acid sequencesdue to amino acid substitutions, deletions, or insertions. In certainaspects, variants can be non-naturally occurring. Non-naturallyoccurring variants can be produced using art-known mutagenesistechniques. Variant polypeptides can comprise conservative ornon-conservative amino acid substitutions, deletions or additions.Derivatives are polypeptides that have been altered so as to exhibitadditional features not found on the original polypeptide. Examplesinclude fusion proteins. Variant polypeptides can also be referred toherein as “polypeptide analogs.” As used herein a “derivative” of apolypeptide can also refer to a subject polypeptide having one or moreamino acids chemically derivatized by reaction of a functional sidegroup. Also included as “derivatives” are those peptides that containone or more derivatives of the twenty standard amino acids. For example,4-hydroxyproline can be substituted for proline; 5-hydroxylysine can besubstituted for lysine; 3-methylhistidine can be substituted forhistidine; homoserine can be substituted for serine; and ornithine canbe substituted for lysine.

A “conservative amino acid substitution” is one in which one amino acidis replaced with another amino acid having a similar side chain.Families of amino acids having similar side chains have been defined inthe art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., asparagine, glutamine, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. In certainembodiments, conservative substitutions in the sequences of thepolypeptides and antibodies of the present disclosure do not abrogatethe binding of the polypeptide or antibody containing the amino acidsequence, to the antigen to which the binding molecule binds. Methods ofidentifying nucleotide and amino acid conservative substitutions whichdo not eliminate antigen-binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al.,Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad.Sci. USA 94:412-417 (1997)).

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA), cDNA, or plasmidDNA (pDNA). A polynucleotide can comprise a conventional phosphodiesterbond or a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The terms “nucleic acid” or “nucleic acidsequence” refer to any one or more nucleic acid segments, e.g., DNA orRNA fragments, present in a polynucleotide.

By an “isolated” nucleic acid or polynucleotide is intended any form ofthe nucleic acid or polynucleotide that is separated from its nativeenvironment. For example, gel-purified polynucleotide, or a recombinantpolynucleotide encoding a polypeptide contained in a vector would beconsidered to be “isolated.” Also, a polynucleotide segment, e.g., a PCRproduct, which has been engineered to have restriction sites for cloningis considered to be “isolated.” Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in a non-native solution such as a buffer or saline.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofpolynucleotides, where the transcript is not one that would be found innature. Isolated polynucleotides or nucleic acids further include suchmolecules produced synthetically. In addition, polynucleotide or anucleic acid can be or can include a regulatory element such as apromoter, ribosome binding site, or a transcription terminator.

As used herein, the term “a non-naturally occurring polynucleotide” orany grammatical variants thereof, is a conditional definition thatexplicitly excludes, but only excludes, those forms of the nucleic acidor polynucleotide that are, or could be, determined or interpreted by ajudge, or an administrative or judicial body, to be“naturally-occurring.”

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it can beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions can be present in a single polynucleotideconstruct, e.g., on a single vector, or in separate polynucleotideconstructs, e.g., on separate (different) vectors. Furthermore, anyvector can contain a single coding region, or can comprise two or morecoding regions, e.g., a single vector can separately encode animmunoglobulin heavy chain variable region and an immunoglobulin lightchain variable region. In addition, a vector, polynucleotide, or nucleicacid can include heterologous coding regions, either fused or unfused toanother coding region. Heterologous coding regions include withoutlimitation, those encoding specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally can include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter can be a cell-specificpromoter that directs substantial transcription of the DNA inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit B-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide can be RNA, for example, in theform of messenger RNA (mRNA), transfer RNA, or ribosomal RNA.

Polynucleotide and nucleic acid coding regions can be associated withadditional coding regions which encode secretory or signal peptides,which direct the secretion of a polypeptide encoded by a polynucleotideas disclosed herein. According to the signal hypothesis, proteinssecreted by mammalian cells have a signal peptide or secretory leadersequence which is cleaved from the mature protein once export of thegrowing protein chain across the rough endoplasmic reticulum has beeninitiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells can have a signal peptidefused to the N-terminus of the polypeptide, which is cleaved from thecomplete or “full length” polypeptide to produce a secreted or “mature”form of the polypeptide. In certain embodiments, the native signalpeptide, e.g., an immunoglobulin heavy chain or light chain signalpeptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, can be used. Forexample, the wild-type leader sequence can be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouse8-glucuronidase.

As used herein, the term “CD20” refers to a membrane protein expressedon the surface of B lymphocytes. The CD20 protein is referred to in theliterature by other names, e.g., B-lymphocyte antigen CD20, B-lymphocytecell-surface antigen B1, Bp35, CVID5, LEU-16, Membrane-spanning4-domains subfamily A member 1, or MS4A2. The amino acid sequence forhuman CD20 (GenBank Accession No. NP_690605.1) is disclosed herein asSEQ ID NO: 50 (Table 2).

Disclosed herein are certain binding molecules, or antigen-bindingfragments, variants, or derivatives thereof. Unless specificallyreferring to full-sized antibodies, the term “binding molecule”encompasses full-sized antibodies as well as antigen-binding subunits,fragments, variants, analogs, or derivatives of such antibodies, e.g.,engineered antibody molecules or fragments that bind antigen in a mannersimilar to antibody molecules, but which use a different scaffold.

As used herein, the term “binding molecule” refers in its broadest senseto a molecule that specifically binds to a target or moleculardeterminant, e.g., an epitope or an antigenic determinant. As describedfurther herein, a binding molecule can comprise one or more “antigenbinding domains” described herein. A non-limiting example of a bindingmolecule is an antibody or fragment thereof that retainsantigen-specific binding.

As used herein, the terms “binding domain” or “antigen binding domain”refer to a region of a binding molecule that is sufficient tospecifically bind to an epitope. For example, an “Fv,” e.g., a variableheavy chain and variable light chain of an antibody, either as twoseparate polypeptide subunits or as a single chain, is considered to bea “binding domain.” Other antigen binding domains include, withoutlimitation, the variable heavy chain (VHH) of an antibody derived from acamelid species, or six immunoglobulin complementarity determiningregions (CDRs) expressed in a fibronectin scaffold. A “binding molecule”as described herein can include one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve or more “antigen binding domains.”

The terms “antibody” and “immunoglobulin” can be used interchangeablyherein. An antibody (or a fragment, variant, or derivative thereof asdisclosed herein) includes at least the variable domain of a heavy chain(for camelid species) or at least the variable domains of a heavy chainand a light chain. Basic immunoglobulin structures in vertebrate systemsare relatively well understood. (See, e.g., Harlow et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988).Unless otherwise stated, the term “antibody” encompasses anythingranging from a small antigen-binding fragment of an antibody to a fullsized antibody, e.g., an IgG antibody that includes two complete heavychains and two complete light chains, an IgA antibody that includes fourcomplete heavy chains and four complete light chains and can include aJ-chain and/or a secretory component, or an IgM antibody that includesten or twelve complete heavy chains and ten or twelve complete lightchains and can include a J-chain.

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4 or α1-α2)).It is the nature of this chain that determines the “isotype” of theantibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulinsubclasses (subtypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, IgA₂, are wellcharacterized and are known to confer functional specialization.Modified versions of each of these immunoglobulins are readilydiscernible to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of this disclosure.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class can be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are expressed, e.g., by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. The basicstructure of certain antibodies, e.g., IgG antibodies, includes twoheavy chain subunits and two light chain subunits covalently connectedvia disulfide bonds to form a “Y” structure, also referred to herein asan “H2L2” structure, or a “binding unit.”

The term “binding unit” is used herein to refer to the portion of abinding molecule, e.g., an antibody or antigen-binding fragment thereof,which corresponds to a standard immunoglobulin structure, e.g., twoheavy chains or fragments thereof and two light chains or fragmentsthereof, or two heavy chains or fragments thereof derived, e.g., from acamelid or condricthoid antibody. In certain aspects, e.g., where thebinding molecule is a bivalent IgG antibody or antigen-binding fragmentthereof, the terms “binding molecule” and “binding unit” are equivalent.In other aspects, e.g., where the binding molecule is an IgA dimer, anIgM pentamer, or an IgM hexamer, the binding molecule comprises two ormore “binding units.” Two in the case of an IgA dimer, or five or six inthe case of an IgM pentamer or hexamer, respectively. A binding unitneed not include full-length antibody heavy and light chains, but willtypically be bivalent, i.e., will include two “antigen binding domains,”as defined below. Certain binding molecules provided in this disclosureare dimeric, pentameric, or hexameric, and include two, five, or sixbivalent binding units that include IgA or IgM constant regions orfragments thereof. As used herein, a binding molecule comprising two ormore binding units, e.g., two, five, or six binding units, can bereferred to as “multimeric.”

The term “native sequence J-chain” or “native J-chain” as used hereinrefers to J-chain of native sequence IgM or IgA antibodies of any animalspecies, including mature human J-chain, the amino acid sequence ofwhich is presented as SEQ ID NO: 49.

The term “modified J-chain” is used herein to refer to variants ofnative sequence J-chain polypeptides comprising a heterologous moiety,e.g., a heterologous polypeptide, e.g., an extraneous binding domainintroduced into the native sequence. The introduction can be achieved byany means, including direct or indirect fusion of the heterologouspolypeptide or other moiety or by attachment through a peptide orchemical linker. The term “modified human J-chain” encompasses, withoutlimitation, a native sequence human J-chain of the amino acid sequenceof SEQ ID NO: 49 or functional fragment thereof modified by theintroduction of a heterologous moiety, e.g., a heterologous polypeptide,e.g., an extraneous binding domain. In certain aspects the heterologousmoiety does not interfere with efficient polymerization of IgM into apentamer or IgA into a dimer and binding of such polymers to a target.Exemplary modified J-chains can be found, e.g., in PCT Publication No.WO 2015/153912, which is incorporated herein by reference in itsentirety.

The terms “valency,” “bivalent,” “multivalent” and grammaticalequivalents, refer to the number of antigen binding domains in givenbinding molecule or binding unit. As such, the terms “bivalent”,“tetravalent”, and “hexavalent” in reference to a given bindingmolecule, e.g., an IgM antibody or fragment thereof, denote the presenceof two antigen binding domains, four antigen binding domains, and sixantigen binding domains, respectively. In a typical IgM-derived bindingmolecule where each binding unit is bivalent, the binding moleculeitself can have 10 or 12 valencies. In a typical IgA-derived bindingmolecule where each binding unit is bivalent, the binding moleculeitself can have 4 valencies. A bivalent or multivalent binding moleculecan be monospecific, i.e., all of the antigen binding domains are thesame, or can be bispecific or multispecific, e.g., where two or moreantigen binding domains are different, e.g., bind to different epitopeson the same antigen, or bind to entirely different antigens.

The term “epitope” includes any molecular determinant capable ofspecific binding to an antibody. In certain aspects, an epitope caninclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl, or sulfonyl, and, in certainaspects, can have three-dimensional structural characteristics, and orspecific charge characteristics. An epitope is a region of a target thatis bound by an antibody.

“Multispecific binding molecules or antibodies” or “bispecific bindingmolecules or antibodies” refer to binding molecules, antibodies, orantigen-binding fragments thereof that have the ability to specificallybind to two or more different epitopes on the same or differenttarget(s). “Monospecific” refers to the ability to bind only oneepitope.

The term “target” is used in the broadest sense to include substancesthat can be bound by a binding molecule. A target can be, e.g., apolypeptide, a nucleic acid, a carbohydrate, a lipid, or other molecule.Moreover, a “target” can, for example, be a cell, an organ, or anorganism that comprises an epitope bound that can be bound by a bindingmolecule.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the variable light (VL) and variable heavy (VH) chainportions determine antigen recognition and specificity. Conversely, theconstant domains of the light chain (CL) and the heavy chain (e.g., CH1,CH2, CH3, or CH4) confer biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, andthe like. By convention the numbering of the constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the antibody. The N-terminal portion is a variableregion and at the C-terminal portion is a constant region; the CH3 (orCH4 in the case of IgM) and CL domains are at the carboxy-terminus ofthe heavy and light chain, respectively.

A “full length IgM antibody heavy chain” is a polypeptide that includes,in N-terminal to C-terminal direction, an antibody heavy chain variabledomain (VH), an antibody constant heavy chain constant domain 1 (CM1 orCμ1), an antibody heavy chain constant domain 2 (CM2 or Cμ2), anantibody heavy chain constant domain 3 (CM3 or Cμ3), and an antibodyheavy chain constant domain 4 (CM4 or Cμ4) that can include a tailpiece.

A “full length IgA antibody heavy chain” is a polypeptide that includes,in N-terminal to C-terminal direction, an antibody heavy chain variabledomain (VH), an antibody constant heavy chain constant domain 1 (CA1 orCal), an antibody heavy chain constant domain 2 (CA2 or Cα2), anantibody heavy chain constant domain 3 (CA3 or C3) that can include atailpiece. The structure of monomeric and secretory IgA is described,e.g., in Woof, J M and Russell, MW, Mucosal Immunology 4:590-597 (2011).

As indicated above, a variable region (i.e., the “antigen bindingdomain”) allows a binding molecule to selectively recognize andspecifically bind epitopes on antigens. That is, the VL domain and VHdomain (or just a VH domain for camelid or condricthoid antibodies(designated as VHH)), or subset of the complementarity determiningregions (CDRs), of a binding molecule, e.g., an antibody, can combine toform the antigen binding domain. More specifically, an antigen bindingdomain can be defined by three CDRs on each of the VH and VL chains (or3 CDRs on a VHH). Certain antibodies form larger structures. Forexample, IgA can form a molecule that includes two H2L2 binding units, aJ-chain, and a secretory component, covalently connected via disulfidebonds; and IgM can form a dimeric, pentameric, or hexameric moleculethat includes two, five, or six H2L2 binding units and optionally aJ-chain covalently connected via disulfide bonds.

The six “complementarity determining regions” or “CDRs” present in anantibody antigen binding domain are short, non-contiguous sequences ofamino acids that are specifically positioned to form the antigen bindingdomain as the antibody assumes its three-dimensional configuration in anaqueous environment. The remainder of the amino acids in the antigenbinding domain, referred to as “framework” regions, show lessinter-molecular variability. The framework regions largely adopt aβ-sheet conformation and the CDRs form loops which connect, and in somecases form part of, the β-sheet structure. Thus, framework regions actto form a scaffold that provides for positioning the CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto its cognate epitope. The amino acids that make up the CDRs and theframework regions, respectively, can be readily identified for any givenheavy or light chain variable region by one of ordinary skill in theart, since they have been defined in various different ways (see,“Sequences of Proteins of Immunological Interest,” Kabat, E., et al.,U.S. Department of Health and Human Services, (1983); and Chothia andLesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated hereinby reference in their entireties).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. These particular regionshave been described, for example, by Kabat et al., U.S. Dept. of Healthand Human Services, “Sequences of Proteins of Immunological Interest”(1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), whichare incorporated herein by reference. The Kabat and Chothia definitionsinclude overlapping or subsets of amino acids when compared against eachother. Nevertheless, application of either definition (or otherdefinitions known to those of ordinary skill in the art) to refer to aCDR of an antibody or variant thereof is intended to be within the scopeof the term as defined and used herein, unless otherwise indicated. Theappropriate amino acids which encompass the CDRs as defined by each ofthe above cited references are set forth below in Table 1 as acomparison. The exact amino acid numbers which encompass a particularCDR will vary depending on the sequence and size of the CDR. Thoseskilled in the art can routinely determine which amino acids comprise aparticular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 CDR Definitions* Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 *Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Immunoglobulin variable domains can also be analyzed, e.g., using theIMGT information system (www://imgt.cines.fr/) (IMGT®/V-Quest) toidentify variable region segments, including CDRs. See, e.g., Brochet,X. et al., Nucl. Acids Res. 36:W503-508 (2008).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless use of the Kabat numbering system is explicitly noted, however,consecutive numbering is used for amino acid sequences in thisdisclosure.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants, or derivatives thereof include, but are not limited to,polyclonal, monoclonal, human, humanized, or chimeric antibodies, singlechain antibodies, epitope-binding fragments, e.g., Fab, Fab′ andF(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library. ScFv moleculesare known in the art and are described, e.g., in U.S. Pat. No.5,892,019.

By “specifically binds,” it is generally meant that a binding molecule,e.g., an antibody or fragment, variant, or derivative thereof binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, a binding molecule is said to“specifically bind” to an epitope when it binds to that epitope, via itsantigen binding domain more readily than it would bind to a random,unrelated epitope. The term “specificity” is used herein to qualify therelative affinity by which a certain binding molecule binds to a certainepitope. For example, binding molecule “A” can be deemed to have ahigher specificity for a given epitope than binding molecule “B,” orbinding molecule “A” can be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof disclosed herein can be said to bind a target antigenwith an off rate (k(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻²sec⁻¹, 5×10⁻³ sec⁻¹, 10⁻³ sec⁻¹, 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹,or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein can be said to bind a targetantigen with an on rate (k(on)) of greater than or equal to 10⁻³ M⁻¹sec⁻¹, 5×10⁻³ M⁻¹ sec⁻¹, 10⁻⁴ M⁻¹ sec⁻¹, 5×10⁻⁴ M⁻¹ sec⁻¹, 10⁻⁵ M⁻¹sec⁻¹, 5×10⁻⁵ M⁻¹ sec⁻¹, 10⁻⁶ M⁻¹ sec⁻¹, or 5×10⁻⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹sec⁻¹.

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof is said to competitively inhibit binding of areference antibody or antigen binding fragment to a given epitope if itpreferentially binds to that epitope to the extent that it blocks, tosome degree, binding of the reference antibody or antigen bindingfragment to the epitope. Competitive inhibition can be determined by anymethod known in the art, for example, competition ELISA assays. Abinding molecule can be said to competitively inhibit binding of thereference antibody or antigen binding fragment to a given epitope by atleast 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with one or more antigen bindingdomains, e.g., of an immunoglobulin molecule. See, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population of antigenbinding domains and an antigen. See, e.g., Harlow at pages 29-34.Avidity is related to both the affinity of individual antigen bindingdomains in the population with specific epitopes, and also the valenciesof the immunoglobulins and the antigen. For example, the interactionbetween a bivalent monoclonal antibody and an antigen with a highlyrepeating epitope structure, such as a polymer, would be one of highavidity. An interaction between a between a bivalent monoclonal antibodywith a receptor present at a high density on a cell surface would alsobe of high avidity.

Binding molecules or antigen-binding fragments, variants or derivativesthereof as disclosed herein can also be described or specified in termsof their cross-reactivity. As used herein, the term “cross-reactivity”refers to the ability of a binding molecule, e.g., an antibody orfragment, variant, or derivative thereof, specific for one antigen, toreact with a second antigen; a measure of relatedness between twodifferent antigenic substances. Thus, a binding molecule is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross-reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, can actually fit better than the original.

A binding molecule, e.g., an antibody or fragment, variant, orderivative thereof can also be described or specified in terms of theirbinding affinity to an antigen. For example, a binding molecule can bindto an antigen with a dissociation constant or K_(D) no greater than5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶ M, 5×10⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 109 M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹5 M.

Antibody fragments including single-chain antibodies or other antigenbinding domains can exist alone or in combination with one or more ofthe following: hinge region, CH1, CH2, CH3, or CH4 domains, J-chain, orsecretory component. Also included are antigen-binding fragments thatcan include any combination of variable region(s) with one or more of ahinge region, CH1, CH2, CH3, or CH4 domains, a J-chain, or a secretorycomponent. Binding molecules, e.g., antibodies, or antigen-bindingfragments thereof can be from any animal origin including birds andmammals. The antibodies can be human, murine, donkey, rabbit, goat,guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region can be condricthoid in origin (e.g.,from sharks). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and can in someinstances express endogenous immunoglobulins and some not, as describedinfra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati etal.

As used herein, the term “heavy chain subunit” includes amino acidsequences derived from an immunoglobulin heavy chain, a bindingmolecule, e.g., an antibody comprising a heavy chain subunit can includeat least one of a VH domain, a CH1 domain, a hinge (e.g., upper, middle,and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4domain, or a variant or fragment thereof. For example, a bindingmolecule, e.g., an antibody or fragment, variant, or derivative thereofcan include, without limitation, in addition to a VH domain: a CH1domain; a CH1 domain, a hinge, and a CH2 domain; a CH1 domain and a CH3domain; a CH1 domain, a hinge, and a CH3 domain; or a CH1 domain, ahinge domain, a CH2 domain, and a CH3 domain. In certain aspects abinding molecule, e.g., an antibody or fragment, variant, or derivativethereof can include, in addition to a VH domain, a CH3 domain and a CH4domain; or a CH3 domain, a CH4 domain, and a J-chain. Further, a bindingmolecule for use in the disclosure can lack certain constant regionportions, e.g., all or part of a CH2 domain. It will be understood byone of ordinary skill in the art that these domains (e.g., the heavychain subunit) can be modified such that they vary in amino acidsequence from the original immunoglobulin molecule.

As used herein, the term “light chain subunit” includes amino acidsequences derived from an immunoglobulin light chain. The light chainsubunit includes at least a VL, and can further include a CL (e.g., Cκor Cλ) domain.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants, or derivatives thereof can be described or specified in termsof the epitope(s) or portion(s) of an antigen that they recognize orspecifically bind. The portion of a target antigen that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target antigen can comprisea single epitope or at least two epitopes, and can include any number ofepitopes, depending on the size, conformation, and type of antigen.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup.

As used herein, the term “chimeric antibody” refers to an antibody inwhich the immunoreactive region or site is obtained or derived from afirst species and the constant region (which can be intact, partial ormodified) is obtained from a second species. In some embodiments thetarget binding region or site will be from a non-human source (e.g.mouse or primate) and the constant region is human.

The term “multispecific antibody, e.g., “bispecific antibody” refers toan antibody that has antigen binding domains for two or more differentepitopes within a single antibody molecule. Other binding molecules inaddition to the canonical antibody structure can be constructed with twobinding specificities. Epitope binding by bispecific or multispecificantibodies can be simultaneous or sequential. Triomas and hybridhybridomas are two examples of cell lines that can secrete bispecificantibodies. Bispecific antibodies can also be constructed by recombinantmeans. (Strohlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry andSnavely, IDrugs. 13:543-9 (2010)). A bispecific antibody can also be adiabody.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more amino acids ineither the CDR or framework regions. In certain aspects entire CDRs froman antibody of known specificity can be grafted into the frameworkregions of a heterologous antibody. Although alternate CDRs can bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, CDRs can also bederived from an antibody of different class, e.g., from an antibody froma different species. An engineered antibody in which one or more “donor”CDRs from a non-human antibody of known specificity are grafted into ahuman heavy or light chain framework region is referred to herein as a“humanized antibody.” In certain aspects not all of the CDRs arereplaced with the complete CDRs from the donor variable region and yetthe antigen binding capacity of the donor can still be transferred tothe recipient variable domains. Given the explanations set forth in,e.g., U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, itwill be well within the competence of those skilled in the art, eitherby carrying out routine experimentation or by trial and error testing toobtain a functional engineered or humanized antibody.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” or “fusion” or othergrammatical equivalents can be used interchangeably. These terms referto the joining together of two more elements or components, by whatevermeans including chemical conjugation or recombinant means. An “in-framefusion” refers to the joining of two or more polynucleotide open readingframes (ORFs) to form a continuous longer ORF, in a manner thatmaintains the translational reading frame of the original ORFs. Thus, arecombinant fusion protein is a single protein containing two or moresegments that correspond to polypeptides encoded by the original ORFs(which segments are not normally so joined in nature.) Although thereading frame is thus made continuous throughout the fused segments, thesegments can be physically or spatially separated by, for example,in-frame linker sequence. For example, polynucleotides encoding the CDRsof an immunoglobulin variable region can be fused, in-frame, but beseparated by a polynucleotide encoding at least one immunoglobulinframework region or additional CDR regions, as long as the “fused” CDRsare co-translated as part of a continuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which amino acids that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide. Aportion of a polypeptide that is “amino-terminal” or “N-terminal” toanother portion of a polypeptide is that portion that comes earlier inthe sequential polypeptide chain. Similarly, a portion of a polypeptidethat is “carboxy-terminal” or “C-terminal” to another portion of apolypeptide is that portion that comes later in the sequentialpolypeptide chain. For example, in a typical antibody, the variabledomain is “N-terminal” to the constant region, and the constant regionis “C-terminal” to the variable domain.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into RNA, e.g., messenger RNA (mRNA), and thetranslation of such mRNA into polypeptide(s). If the final desiredproduct is a biochemical, expression includes the creation of thatbiochemical and any precursors. Expression of a gene produces a “geneproduct.” As used herein, a gene product can be either a nucleic acid,e.g., a messenger RNA produced by transcription of a gene, or apolypeptide that is translated from a transcript. Gene productsdescribed herein further include nucleic acids with post transcriptionalmodifications, e.g., polyadenylation, or polypeptides with posttranslational modifications, e.g., methylation, glycosylation, theaddition of lipids, association with other protein subunits, proteolyticcleavage, and the like.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt or slow the progression of an existingdiagnosed pathologic condition or disorder. Terms such as “prevent,”“prevention,” “avoid,” “deterrence” and the like refer to prophylacticor preventative measures that prevent the development of an undiagnosedtargeted pathologic condition or disorder. Thus, “those in need oftreatment” can include those already with the disorder; those prone tohave the disorder; and those in whom the disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows,bears, and so on.

As used herein, phrases such as “a subject that would benefit fromtherapy” and “an animal in need of treatment” includes subjects, such asmammalian subjects, that would benefit from administration of a bindingmolecule such as an antibody, comprising one or more antigen bindingdomains. Such binding molecules, e.g., antibodies, can be used, e.g.,for diagnostic procedures and/or for treatment or prevention of adisease.

IgM Binding Molecules

IgM is the first immunoglobulin produced by B cells in response tostimulation by antigen and is present at around 1.5 mg/ml in serum witha half-life of 5 days. IgM is a dimeric, pentameric, or hexamericmolecule. An IgM binding unit includes two light and two heavy chains.While IgG contains three heavy chain constant domains (CH1, CH2 andCH3), the heavy (p) chain of IgM additionally contains a fourth constantdomain (CH4), that includes a C-terminal “tailpiece.” The human IgMconstant region typically comprises the amino acid sequence SEQ ID NO:47. The human Cμ1 region ranges from about amino acid 5 to about aminoacid 102 of SEQ ID NO: 47; the human Cμ2 region ranges from about aminoacid 114 to about amino acid 205 of SEQ ID NO: 47, the human Cμ3 regionranges from about amino acid 224 to about amino acid 319 of SEQ ID NO:47, the Cμ4 region ranges from about amino acid 329 to about amino acid430 of SEQ ID NO: 47, and the tailpiece ranges from about amino acid 431to about amino acid 453 of SEQ ID NO: 47 (Table 2).

Five IgM binding units can form a complex with an additional smallpolypeptide chain (the J-chain) to form an IgM antibody. The humanJ-chain comprises the amino acid sequence SEQ ID NO: 49 (Table 2). Atypical IgM pentamer is depicted in FIG. 2. Without the J-chain, IgMbinding units typically assemble into a hexamer. A typical IgM hexameris depicted in FIG. 2. While not wishing to be bound by theory, theassembly of IgM binding units into a hexameric or pentameric bindingmolecule is thought to involve the Cμ3 and Cμ4 domains. Accordingly, ahexameric or pentameric binding molecule provided in this disclosuretypically includes IgM constant regions that include at least the Cμ3and Cμ4 domains. A comparison of IgG antibodies and IgM antibodies bynon-reducing polyacrylamide gel electrophoresis is shown in FIG. 3.

An IgM heavy chain constant region can additionally include a Cμ2 domainor a fragment thereof, a Cμ1 domain or a fragment thereof, and/or otherIgM heavy chain domains. In certain aspects, a binding molecule asprovided herein can include a complete IgM heavy (p) chain constantdomain (e.g., SEQ ID NO: 47), or a variant, derivative, or analogthereof.

Pentameric or Hexameric CD20 Binding Molecules

This disclosure provides a hexameric or pentameric binding molecule,i.e., a binding molecule with five or six “binding units” as definedherein, that can specifically bind to CD20, e.g., human CD20. A bindingmolecule as provided herein can possess improved binding characteristicsor biological activity as compared to a binding molecule composed of asingle binding unit, e.g., a bivalent IgG antibody. In certain aspects,the disclosure provides a pentameric or hexameric binding moleculecomprising five or six bivalent binding units, respectively, where eachbinding unit includes two IgM heavy chain constant regions or fragmentsthereof. In certain aspects, the two IgM heavy chain constant regionsare human heavy chain constant regions.

Where the binding molecule provided herein is pentameric, the bindingmolecule can further comprise a J-chain, or functional fragment thereof,or variant thereof. In certain aspects, the J-chain is a modifiedJ-chain comprising a heterologous moiety or one or more heterologousmoieties, e.g., a heterologous polypeptide sequence, e.g., an extraneousbinding domain introduced into the native sequence. In certain aspectsthe extraneous binding domain specifically binds to CD3, e.g., CD3ε. Incertain aspects the modified J-chain comprises V15J (SEQ ID NO: 64) orJ15V (SEQ ID NO: 66).

An IgM heavy chain constant region can include one or more of a Cμ1domain, a Cμ2 domain, a Cμ3 domain, and/or a Cμ4 domain, provided thatthe constant region can serve a desired function in the bindingmolecule, e.g., associate with second IgM constant region to form anantigen binding domain, or associate with other binding units to form ahexamer or a pentamer. In certain aspects the two IgM heavy chainconstant regions or fragments thereof within an individual binding uniteach comprise a Cμ3 domain or fragment thereof, a Cμ4 domain or fragmentthereof, a tailpiece (TP) or fragment thereof, or any combination of aCμ3 domain a Cμ domain, and a TP or fragment thereof. In certain aspectsthe two IgM heavy chain constant regions or fragments thereof within anindividual binding unit each further comprise a Cμ2 domain or fragmentthereof, a Cμ1 domain or fragment thereof, or a Cμ1 domain or fragmentthereof and a Cμ2 domain or fragment thereof.

In certain aspects each of the two IgM heavy chain constant regions in agiven binding unit is associated with an antigen binding domain, forexample an Fv portion of an antibody, e.g., a VH and a VL of a human ormurine antibody. In a binding molecule as provided herein at least oneantigen binding domain of the binding molecule is an anti-CD20 antigenbinding domain, i.e., an antigen binding domain that can specificallybind to CD20, e.g., human CD20.

IgA Binding Molecules

IgA plays a role in mucosal immunity and comprises about 15% of totalimmunoglobulin produced. IgA is a monomeric or dimeric molecule. An IgAbinding unit typically includes two light and two heavy chains. IgAcontains three heavy chain constant domains (Cα1, Cα2 and Cα3), andincludes a C-terminal “tailpiece.” Human IgA has two subtypes, IgA1 andIgA2. The human IgA1 constant region typically comprises the amino acidsequence SEQ ID NO: 59. The human Cα1 region ranges from about aminoacid 6 to about amino acid 98 of SEQ ID NO: 59; the human Cα2 regionranges from about amino acid 125 to about amino acid 220 of SEQ ID NO:59, the human Cα3 region ranges from about amino acid 228 to about aminoacid 330 of SEQ ID NO: 59, and the tailpiece ranges from about aminoacid 331 to about amino acid 352 of SEQ ID NO: 59 (Table 2). The humanIgA2 constant region typically comprises the amino acid sequence SEQ IDNO: 60. The human Cα1 region ranges from about amino acid 6 to aboutamino acid 98 of SEQ ID NO: 60 (Table 2); the human Cα2 region rangesfrom about amino acid 112 to about amino acid 207 of SEQ ID NO: 60, thehuman Cα3 region ranges from about amino acid 215 to about amino acid317 of SEQ ID NO: 60, and the tailpiece ranges from about amino acid 318to about amino acid 340 of SEQ ID NO: 60.

Two IgA binding units can form a complex with two additional polypeptidechains, the J-chain (SEQ ID NO: 49) and the secretory component (SEQ IDNO: 62) to form a secretory IgA (sIgA) antibody. While not wishing to bebound by theory, the assembly of IgA binding units into a dimeric sIgAbinding molecule is thought to involve the Cα3 and tailpiece domains.Accordingly, a dimeric sIgA binding molecule provided in this disclosuretypically includes IgA constant regions that include at least the Cα3and tailpiece domains.

An IgA heavy chain constant region can additionally include a Cα2 domainor a fragment thereof, a Cα1 domain or a fragment thereof, and/or otherIgA heavy chain domains. In certain aspects, a binding molecule asprovided herein can include a complete IgA heavy (a) chain constantdomain (e.g., SEQ ID NO: 59 or SEQ ID NO: 60), or a variant, derivative,or analog thereof.

Dimeric CD20 Binding Molecules

This disclosure provides a dimeric binding molecule, e.g., a bindingmolecule with two IgA “binding units” as defined herein, which canspecifically bind to CD20, e.g., human CD20. A dimeric binding moleculeas provided herein can possess improved binding characteristics orbiological activity as compared to a binding molecule composed of asingle binding unit, e.g., a bivalent IgG antibody. For example, an IgAbinding molecule can reach mucosal sites providing greater tissuedistribution for the binding molecules provided herein. In certainaspects, the disclosure provides a dimeric binding molecule comprisingtwo bivalent binding units, where each binding unit includes two IgAheavy chain constant regions or fragments thereof. In certain aspects,the two IgA heavy chain constant regions are human heavy chain constantregions.

A dimeric IgA binding molecule as provided herein can further comprise aJ-chain, or fragment thereof, or variant thereof. A dimeric IgA bindingmolecule as provided herein can further comprise a secretory component,or fragment thereof, or variant thereof. In certain aspects, the J-chainis a modified J-chain comprising a heterologous moiety or one or moreheterologous moieties, e.g., a heterologous polypeptide, e.g., anextraneous binding domain introduced into the native sequence. Incertain aspects the extraneous binding domain specifically binds to CD3,e.g., CD3ε. In certain aspects the modified J-chain comprises V15J (SEQID NO: 64) or J15V (SEQ ID NO: 66).

An IgA heavy chain constant region can include one or more of a Cα1domain, a Cα2 domain, and/or a Cα3 domain, provided that the constantregion can serve a desired function in the binding molecule, e.g.,associate with a light chain constant region to facilitate formation ofan antigen binding domain, or associate with another IgA binding unit toform a dimeric binding molecule. In certain aspects the two IgA heavychain constant regions or fragments thereof within an individual bindingunit each comprise a C3 domain or fragment thereof, a tailpiece (TP) orfragment thereof, or any combination of a C3 domain, a TP, or fragmentthereof. In certain aspects the two IgA heavy chain constant regions orfragments thereof within an individual binding unit each furthercomprise a Cα2 domain or fragment thereof, a Cα1 domain or fragmentthereof, or a Cα1 domain or fragment thereof and a Cα2 domain orfragment thereof.

In certain aspects each of the two IgA heavy chain constant regions in agiven antigen binding domain is associated with an antigen bindingdomain, for example an Fv portion of an antibody, e.g., a VH and a VL ofa human or murine antibody. In a binding molecule as provided herein atleast one antigen binding domain of the binding molecule can be a CD20antigen binding domain, e.g., a human CD20 antigen binding domain.

Modified J-Chains

In certain aspects CD20 binding molecules provided herein can bebispecific, incorporating a modified J-chain. As provided herein and inPCT Publication No. WO 2015/153912, a modified J-chain can comprise aheterologous moiety, e.g., a heterologous polypeptide, e.g., anextraneous binding domain, which can include, for example, a polypeptidebinding domain capable of specifically binding to a target. The bindingdomain can be, for example, an antibody or antigen-binding fragmentthereof, an antibody-drug conjugate or antigen-binding fragment thereof,or an antibody-like molecule. A polypeptide binding domain can beintroduced into a J-chain by appropriately selecting the location andtype of addition (e.g. direct or indirect fusion, chemical tethering,etc.).

In certain aspects, the binding domain can be an antibody or anantigen-binding fragment of an antibody, including monospecific,bispecific, and multi-specific antibodies and antibody fragments. Theantibody fragment can be, without limitation, a Fab fragment, a Fab′fragment, a F(ab′)₂ fragment, an scFv, (scFv)₂ fragment, single-chainantibody molecules, minibodies, or multispecific antibodies formed fromantibody fragments. In certain aspects, the antibody fragment is a scFv.

In other aspects, the binding domain can be an antibody-like molecule,for example, a human domain antibody (dAb), Dual-Affinity Re-Targeting(DART) molecule, a diabody, a di-diabody, dual-variable domain antibody,a Stacked Variable Domain antibody, a Small Modular ImmunoPharmaceutical (SMIP), a Surrobody, a strand-exchange engineered domain(SEED)-body, or TandAb.

The binding domain can be introduced into the native J-chain sequence atany location that allows the binding of the binding domain to itsbinding target without interfering with the binding of the recipient IgMor IgA molecule to its binding target or binding targets or the abilityof the J-chain to effectively incorporated into an IgA dimer or an IgMpentamer. In certain aspects the binding domain can be inserted at ornear the C-terminus, at or near the mature N-terminus (i.e., amino acidnumber 23 of SEQ ID NO: 49 following cleavage of the signal peptide) orat an internal location that, based on the three-dimensional structureof the J-chain is accessible. In certain aspects, the binding domain canbe introduced into the native sequence J-chain without about 10 residuesfrom the C-terminus or without about 10 amino acid residues from themature N-terminus, of the human J-chain of SEQ ID NO: 49. In anotheraspect, the binding domain can be introduced into the native sequencehuman J-chain of SEQ ID NO: 49 in between cysteine residues 114 and 123of SEQ ID NO: 49, or at an equivalent location of another nativesequence J-chain. In a further aspect, the binding domain can beintroduced into a native sequence J-chain, such as a J-chain of SEQ IDNO: 49, at or near a glycosylation site. In certain aspects, the bindingdomain can be introduced into the native sequence human J-chain of SEQID NO: 49 within about 10 amino acid residues from the C-terminus.

Introduction can be accomplished by direct or indirect fusion, i.e. bythe combination of the J-chain and binding domain in one polypeptidechain by in-frame combination of their coding nucleotide sequences, withor without a peptide linker. The peptide linker (indirect fusion), ifused, can be about 1 to 50, or about 1 to 40, or about 1 to 30, or about1 to 20, or about 1 to 10, or about 10 to 20 amino acids in length, andcan be present at one or both ends of the binding domain to beintroduced into the J-chain sequence. In certain aspects, the peptidelinker is about 10 to 20, or 10 to 15 amino acids long. In certainaspects the peptide linker is 15 amino acids long. In certain aspectsthe peptide linker is (GGGGS)₃ (SEQ ID NO: 67).

It is also possible to introduce more than one heterologous polypeptide,e.g., more than one binding domain, into a J-chain.

The modified J-chain can be produced by well-known techniques ofrecombinant DNA technology, by expressing a nucleic acid encoding themodified J-chain in a suitable prokaryotic or eukaryotic host organism.

The modified J-chain can also be co-expressed with the heavy and lightchains of the recipient IgM or IgA binding molecules as describedelsewhere herein. The recipient binding molecule, prior to modifiedJ-chain incorporation, can be monospecific, bispecific ormulti-specific, e.g., a monospecific, bispecific, or multispecific IgAor IgM antibody. Bispecific and multi-specific IgM and IgA bindingmolecules, including antibodies, are described, for example, in U.S.Application Ser. Nos. 61/874,277 and 61/937,984, the entire contents ofwhich are hereby expressly incorporated by reference.

In certain aspects, an anti-CD20 IgM or IgA binding molecule asdescribed herein can include a modified J-chain with binding specificityfor an immune effector cell, such as a T-cell, NK-cell, a macrophage, ora neutrophil. In certain aspects the effector cell is a T-cell and thebinding target is CD3 (discussed below). By activating and redirectingeffector cells, e.g. effector T-cells, to CD20-expressing B cells, e.g.,malignant B cells, a bispecific anti-CD20 x anti-CD3 IgM or IgA bindingmolecule as provided herein can produce an enhanced immune responseagainst the target, the response comprising, e.g., complement-mediatedcytotoxicity and/or antibody dependent cellular cytotoxicity (ADCC),thereby further increasing potency and efficacy. In certain aspects, abispecific anti-CD20 x anti-CD3 IgM or IgA binding molecule as providedherein comprising a modified J-chain can be used for the treatment ofB-cell related cancers.

In the case of T-cells, cluster of differentiation 3 (CD3) is amultimeric protein complex, known historically as the T3 complex, and iscomposed of four distinct polypeptide chains (ε, γ, δ, ζ) that assembleand function as three pairs of dimers (εγ, εδ, ζζ). The CD3 complexserves as a T-cell co-receptor that associates non-covalently with theT-cell receptor (TCR). Components of this CD3 complex, especially CD3ε,can be targets for a modified J-chain of a bispecific IgM or IgA bindingmolecule provided herein.

In certain aspects, a bispecific anti-CD20 x anti-CD3 IgM or IgA bindingmolecule binds to CD20 via the antibody binding domains, while theJ-chain is modified to bind to CD3ε.

In certain aspects the anti-CD3ε binding domain of a modified J-chainprovided herein is a scFv. The anti CD3ε scFv can be fused at or nearthe N-terminus of the J-chain, or at or near the C-terminus of theJ-chain either directly or indirectly with a synthetic linker introducedin between the scFv and the J-chain sequences, e.g., a (GGGGS)₃ linker(SEQ ID NO: 67). In certain aspects the scFv comprises the VH and VLregions of visilizumab (Nuvion). In certain aspects the modified J-chaincomprises a scFv comprising the VH of visilizumab, a (GGGGS)₃ linker,and the VL of visilizumab.

In certain aspects the modified J-chain comprises a scFv of visilizumabfused to the N-terminus of the human J-chain through a 15-amino acid(GGGGS)₃ linker, a modified J-chain referred to herein as V15J. V15J canfurther include a signal peptide to facilitate transport and assemblyinto an IgM or IgA binding molecules. The mature V15J protein ispresented as SEQ ID NO: 64, the precursor version, comprising a 19-aminoacid-immunoglobulin heavy chain signal peptide is presented as SEQ IDNO: 63. In certain aspects the modified J-chain comprises a scFv ofvisilizumab fused to the C-terminus of the human J-chain through a15-amino acid (GGGGS)₃ linker, a modified J-chain referred to herein asJ15V. J15V can further include a signal peptide to facilitate transportand assembly into an IgM or IgA binding molecules. The mature J15Vprotein is presented as SEQ ID NO: 65, the precursor version, comprisingthe 22-amino acid-human J-chain signal peptide is presented as SEQ IDNO: 66. In certain aspects, other signal peptides can be used. Selectionand inclusion of suitable signal peptides to facilitate expression,secretion, and incorporation of a modified J-chain into an anti-CD20 IgMor IgA binding molecule as provided herein is well within thecapabilities of a person of ordinary skill in the art.

CD20 Binding Domains

In certain aspects the CD20 antigen binding domain comprises siximmunoglobulin complementarity determining regions HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3, wherein at least one, at least two, at leastthree, at least four, at least five, or at least six CDRs are related tothe corresponding CDRs of 1.5.3 disclosed in U.S. Patent Publication No.2007-0014720. The CD20 antigen binding domain can include an HCDR1comprising the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 39with one, two, three, four, or five single amino acid substitutions,e.g., one or two single amino acid substitutions. The CD20 antigenbinding domain can include an HCDR2 comprising the amino acid sequenceof SEQ ID NO: 40 or SEQ ID NO: 40 with one, two, three, four, or fivesingle amino acid substitutions, e.g., one or two single amino acidsubstitutions. The CD20 antigen binding domain can include an HCDR3comprising the amino acid sequence of SEQ ID NO: 41, or SEQ ID NO: 41with one, two, three, four, or five single amino acid substitutions,e.g., one or two single amino acid substitutions. The CD20 antigenbinding domain can include an LCDR1 comprising the amino acid sequenceof SEQ ID NO: 43, or SEQ ID NO: 43 with one, two, three, four, or fivesingle amino acid substitutions, e.g., one or two single amino acidsubstitutions. The CD20 antigen binding domain can include an LCDR2comprising the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 44with one, two, three, four, or five single amino acid substitutions,e.g., one or two single amino acid substitutions. The CD20 antigenbinding domain can include an LCDR3 comprising the amino acid sequenceof SEQ ID NO: 45 or SEQ ID NO: 45 with one, two, three, four, or fivesingle amino acid substitutions, e.g., one or two single amino acidsubstitutions. The CD20 antigen binding domain can include any one, anytwo, any three, any four, any five or all six of the CDR amino acidsequences as described above. In certain aspects the CD20 antigenbinding domain includes an HCDR1 comprising the amino acid sequence SEQID NO: 39, an HCDR2 comprising the amino acid sequence SEQ ID NO: 40, anHCDR3 comprising the amino acid sequence SEQ ID NO: 41, an LCDR1comprising the amino acid sequence SEQ ID NO: 43, an LCDR2 comprisingthe amino acid sequence SEQ ID NO: 44, and an LCDR3 comprising the aminoacid sequence SEQ ID NO: 45.

In certain aspects the CD20 antigen binding domain comprises an antibodyheavy chain variable region (VH) and an antibody light chain variableregion (VL), wherein the VH region, the VL region, or both the VH and VLregions are related to the corresponding VH and VL of 1.5.3 disclosed inU.S. Patent Publication No. 2007-0014720. In certain aspects the VH cancomprise an amino acid sequence at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% or 100% identical to SEQ ID NO: 38. In certain aspects the VL cancomprise an amino acid sequence at least at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or 100% identical to SEQ ID NO: 42. In certain aspects the VHcomprises the amino acid sequence SEQ ID NO: 38 and the VL comprises theamino acid sequence SEQ ID NO: 42.

While a variety of different dimeric, pentameric, or hexameric bindingmolecules can be contemplated by a person of ordinary skill in the artbased on this disclosure, and as such are included in this disclosure,in certain aspects, a binding molecule as described above is provided inwhich each binding unit comprises two IgA or IgM heavy chains eachcomprising a VH situated amino terminal to the IgA or IgM constantregion or fragment thereof, and two immunoglobulin light chains eachcomprising a VL situated amino terminal to an immunoglobulin light chainconstant region.

Moreover, in certain aspects, at least one binding unit of the bindingmolecule, or at least two, at least three, at least four, at least five,or at least six binding units of the binding molecule, comprises orcomprise two of the CD20 antigen binding domains as described above. Incertain aspects the two CD20 antigen binding domains in the binding unitof the binding molecule, or the two, three, four, five, or six bindingunits of the binding molecule, can be different from each other, or theycan be identical.

In certain aspects, the two IgM heavy chains within the one, two, three,four, five, or six binding unit(s) of the binding molecule areidentical. In certain aspects, two identical IgM heavy chains within atleast one binding unit, or within at least two, at least three, at leastfour, at least five, or at least six binding units of the bindingmolecule comprise the amino acid sequence SEQ ID NO: 56.

In certain aspects, the two light chains within the one, two, three,four, five, or six binding unit(s) of the binding molecule areidentical. In certain aspects, two identical light chains within atleast one binding unit, or within at least two, at least three, at leastfour, at least five, or at least six binding units of the bindingmolecule are kappa light chains, e.g., human kappa light chains, orlambda light chains, e.g., human lambda light chains. In certainaspects, two identical light chains within at least one binding unit, orwithin at least two, at least three, at least four, at least five, or atleast six binding units of the binding molecule each comprise the aminoacid sequence SEQ ID NO: 58.

In certain aspects at least one, at least two, at least three, at leastfour, at least five, or at least six binding units of a dimeric,pentameric, or hexameric binding molecule provided by this disclosurescomprises or each comprise two identical IgM heavy chains eachcomprising the amino acid sequence SEQ ID NO: 56, and two identicallight chains each comprising the amino acid sequence SEQ ID NO: 58.According to this aspect, the CD20 antigen binding domains in the one,two, three, four, five, or six binding unit(s) of the binding molecule,can be identical. Further according to this aspect, a dimeric,pentameric, or hexameric binding molecule as provided herein cancomprise at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,at least ten, at least eleven, or at least twelve copies of a CD20antigen binding domain as described above. In certain aspects at leasttwo, at least three, at least four, at least five, or at least six ofthe binding units can be identical and, in certain aspects the bindingunits can comprise identical antigen binding domains, e.g., at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten, at leasteleven, or at least twelve CD20 antigen binding domains can beidentical. In certain aspects the identical CD20 antigen binding domaincan comprise a VH with the amino acid sequence SEQ ID NO: 38, and a VLwith the amino acid sequence SEQ ID NO: 42.

In certain aspects, a dimeric, pentameric, or hexameric CD20 bindingmolecule as provided herein can possess advantageous structural orfunctional properties compared to other binding molecules. For example,the dimeric, pentameric, or hexameric CD20 binding molecule can possessimproved activity in a biological assay, either in vitro or in vivo,than a corresponding binding molecule, e.g., an IgG1 1.5.3 as disclosedin U.S. Patent Publication No. 2007-0014720. Biological assays include,but are not limited to, complement-dependent cellular cytotoxicity (CDC)and antibody dependent cellular cytotoxicity (ADCC).

In certain aspects a dimeric, pentameric, or hexameric binding moleculeas provided herein can direct complement-mediated, T-cell-mediated, orboth complement-mediated and T-cell-mediated killing of aCD-20-expressing cell, e.g., a CD20-expressing B cell, at higher potencythan an equivalent amount of a monospecific, bivalent IgG1 antibody orfragment thereof that specifically binds to the same CD20 epitope as theCD20 antigen binding domain, e.g., an IgG1 version of 1.5.3 comprisinghuman IgG1 and a VH with the amino acid sequence SEQ ID NO: 38 and a VLwith the amino acid sequence SEQ ID NO: 42. In certain aspects adimeric, pentameric, or hexameric binding molecule as provided hereincan direct complement-mediated, T-cell-mediated, or bothcomplement-mediated and T-cell-mediated killing of a CD20-expressingcell, e.g., a CD20-expressing B cell at higher potency than anequivalent amount of monospecific, bivalent CD20 monoclonal antibody orfragment thereof, where the antibody is, or comprises the same VH and VLregions as, e.g., rituximab (Genentech), ofatumumab (Glaxo SmithKline),veltuzumab (Takeda), ocaratuzumab (Lilly), tositumumab (GlaxoSmithKline), or obinutumumab (Roche/Genentech).

By “potency” is meant the least amount of a given binding moleculenecessary to achieve a given biological result, e.g., killing of 50% ofthe cells in a given assay, e.g., a CDC or ADCC assay (ICS). Potency canbe expressed as a curve in which % survival of cells is on the Y axis,and binding molecule concentration (in, e.g., μg/ml or μM) is on the Xaxis.

In certain aspects CDC can be measured in vitro, and the CD20-expressingcell can be an immortalized cell line, e.g., a B-cell lymphoma cellline, e.g., a Ramos cell line, a Raji cell line, a Daudi cell line, aNamalwa cell line, a Granta cell line, a Z138 cell line, a DoHH2 cellline, or a DB cell line. Similar cell lines are known and are easilyobtained by a person of ordinary skill in the art.

In certain aspects, CDC can be measured or demonstrated in vitro or invivo, and the CD-20-expressing cell line is a malignant B cell obtainedfrom, or in, a subject, e.g., a human patient, with cancer, e.g., aB-cell related lymphoma, leukemia, or myeloma. In certain aspects thecancer is minimally responsive or non-responsive to conventionaltherapy, e.g., chemotherapy, or monoclonal antibody therapy with one ormore of, e.g., rituximab (Genentech), ofatumumab (Glaxo SmithKline),veltuzumab (Takeda), ocaratuzumab (Lilly), tositumumab (GlaxoSmithKline), or obinutumumab (Roche/Genentech). In certain aspects adimeric, pentameric, or hexameric binding molecule comprising any one ormore of the binding domains described herein, e.g., in Table 5, isprovided.

In certain aspects, ADCC can be measured in vitro through T-cellactivation assays, e.g., by co-culturing CD20-expressing B-cells andengineered CD3-expression T-cells in the presence of a bispecificanti-CD20 x anti-CD3 IgM binding molecule as provided herein, andmeasuring T-cell activation through cytokine release, target cell lysis,or other detection method. In certain aspects ADCC can be measuredthrough T-cell directed B-cell killing. In certain aspects theCD20-expressing cell can be an immortalized cell line, e.g., a B-celllymphoma cell line, e.g., a Ramos cell line, a Raji cell line, a Daudicell line, a Namalwa cell line, a Granta cell line, a Z138 cell line, aDoHH2 cell line, or a DB cell line. Similar cell lines are known and areeasily obtained by a person of ordinary skill in the art. In certainaspects the CD20-expressing cell line can be derived from a patientsuffering from a B-cell related cancer.

In certain aspects, the totality of killing of CD20+ cells, e.g., byCDC, ADCC, and other modes of killing, e.g., apoptosis, can be tested invitro in an assay using whole blood that includes both T-cells andcomplement.

In certain aspects, e.g., where the binding molecule is a pentamericbinding molecule comprising five identical binding units each comprisingtwo identical CD20 binding domains with, e.g., the VH and VL of 1.5.3,tested in a CDC assay using, e.g., the CD20-expressing Raji cell line,the binding molecule can direct complement mediated killing with an IC₅₀at least one-fold, at least two-fold, at least three-fold, at leastfour-fold, at least five-fold, at least ten-fold, at least 20-fold, atleast thirty-fold, at least forty-fold, at least 50-fold, at least100-fold, at least 150-fold, at least 200-fold or more lower than theIC₅₀ of an equivalent amount of the monospecific bivalent IgG1 antibody,e.g., 1.5.3 or rituximab, as measured, e.g., in μg/ml. In certainaspects, where the CD20-expressing cell is a Ramos cell line, thebinding molecule can direct complement-mediated killing with an IC₅₀ atleast ten-fold, at least 20-fold, at least 30-fold, at least 40-fold, atleast 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, atleast 90-fold, or at least 100-fold lower than the IC₅₀ of an equivalentamount of the monospecific bivalent IgG1 antibody, as measured, e.g., asmolar equivalents.

In certain aspects, e.g., a pentameric binding molecule comprising fiveidentical binding units each comprising two identical binding domainswith, e.g., the VH and VL of 1.5.3, or the VH and VL of rituximab, plusa wild-type or modified J-chain as provided herein can exhibit increasedpotency in a CDC assay performed in cells exhibiting lower CD20expression levels. For example, rituximab-derived anti-human CD20 IgM+Jor 1.5.3 anti-CD20 IgM+J, tested in a CDC assay using theCD20-expressing DoHH2 (CD20 high expression) and Z138 (CD20 lowexpression) cell lines, can direct complement-mediated killing with anIC₅₀ at least one-fold, at least two-fold, at least three-fold, at leastfour-fold, at least five-fold, at least ten-fold, at least 20-fold, atleast thirty-fold, at least forty-fold, at least 50-fold, at least100-fold, at least 150-fold, at least 200-fold or more lower than theIC₅₀ of an equivalent amount of the monospecific bivalent IgG1 antibodyequivalents, e.g., rituximab (IgG), or 1.5.3 (IgG1), as measured, e.g.,as molar equivalents. In certain aspects, rituximab-derived anti-humanCD20 IgM+J and 1.5.3 anti-CD20 IgM+J, tested in a CDC assay using theZ138 (CD20 low expression) cell line, can direct complement-mediatedkilling of the cell line under conditions where 50% killing (EC₅₀) withthe equivalent IgG molecules cannot be achieved even at a concentrationof 100 nM.

In certain aspects, a bispecific pentameric binding molecule comprisingfive identical binding units each comprising two identical CD20 bindingdomains with, e.g., the VH and VL of 1.5.3, or the VH and VL ofrituximab, plus a modified J-chain capable of binding to human CD3,e.g., V15J or J15V as provided herein, can exhibit increased potency inan ADCC assay. For example, rituximab-derived anti-human CD20 IgM+V15Jor J15V, or 1.5.3 anti-CD20 IgM+V15J or J15V, tested in a T-cellactivation assay, e.g., using the CD20-expressing DB cell lineco-cultured with engineered Jurkat T-cells, can facilitate T-cellmediated killing with an IC₅₀ at least one-fold, at least two-fold, atleast three-fold, at least four-fold, at least five-fold, at leastten-fold, at least 20-fold, at least thirty-fold, at least forty-fold,at least 50-fold, at least 100-fold, at least 150-fold, at least200-fold or more lower than the IC₅₀ of an equivalent amount of amonovalent bispecific binding molecule that binds B-cells and T-cells,e.g., a bispecific anti-CD19 (monovalent) x anti-CD3 (monovalent)molecule blinatumomab.

In certain aspects, a monospecific or bispecific pentameric bindingmolecule comprising five identical binding units each comprising twoidentical binding domains with, e.g., the VH and VL of 1.5.3, or the VHand VL of rituximab, plus a wild-type J-chain or a modified J-chaincapable of binding to human CD3, e.g., V15J or J15V as provided herein,can exhibit increased potency in a whole-blood in vitro cytotoxicityassay. For example, 1.5.3 anti-CD20 IgM+V15J or J15V, or 1.5.3 anti-CD20IgM+J, tested in a KILR™ in vitro cytotoxicity assay using theCD20-expressing KILR™ ARH-77 cell line co-cultured with Hirudinanti-coagulated human blood can achieve killing of the KILR™ ARH-77 cellline with an IC₅₀ at least one-fold, at least two-fold, at leastthree-fold, at least four-fold, at least five-fold, at least ten-fold,at least 20-fold, at least thirty-fold, at least forty-fold, at least50-fold, at least 100-fold, at least 150-fold, at least 200-fold or morelower than the IC₅₀ of an equivalent amount of a monospecific bivalentanti-CD20 binding molecule, e.g., 1.5.3 IgG, or a monovalent bispecificbinding molecule that binds B-cells and T-cells, e.g., a bispecificanti-CD19 (monovalent) x anti-CD3 (monovalent) molecule blinatumomab.

In certain aspects, a monospecific or bispecific pentameric bindingmolecule comprising five identical binding units each comprising twoidentical binding domains with, e.g., the VH and VL of 1.5.3, or the VHand VL of rituximab, plus a wild-type J-chain or a modified J-chaincapable of binding to human CD3, e.g., V15J or J15V as provided herein,can exhibit significant B-cell killing in vivo, for example in ahumanized mouse model as described elsewhere herein.

Polynucleotides, Vectors, and Host Cells

The disclosure further provides a polynucleotide, e.g., an isolated,recombinant, and/or non-naturally-occurring polynucleotide, comprising anucleic acid sequence that encodes a polypeptide subunit of the dimeric,pentameric, or hexameric binding molecule as described above. By“polypeptide subunit” is meant a portion of a binding molecule, bindingunit, or antigen binding domain that can be independently translated.Examples include, without limitation, an antibody variable domain, e.g.,a VH or a VL, a single chain Fv, an antibody heavy chain, an antibodylight chain, an antibody heavy chain constant region, an antibody lightchain constant region, and/or any fragment thereof.

In certain aspect, the polypeptide subunit can comprise an IgA or IgMheavy chain constant region and at least the antibody VH portion of theCD20 antigen binding domain. In certain aspects the polynucleotide canencode a polypeptide subunit comprising a human IgA or IgM constantregion or fragment thereof fused to the C-terminal end of a VH, wherethe VH comprises an HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprisesthe amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 39 with one, two,three, four, or five single amino acid substitutions, e.g., one or twosingle amino acid substitutions; the HCDR2 comprises the amino acidsequence of SEQ ID NO: 40 or SEQ ID NO: 40 with one, two, three, four,or five single amino acid substitutions, e.g., or two single amino acidsubstitutions; the HCDR3 comprises the amino acid sequence of SEQ ID NO:41, SEQ ID NO: 41 with one, two, three, four, or five single amino acidsubstitutions, e.g., one or two single amino acid substitutions; or theVH comprises an amino acid sequence at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% or 100% identical to SEQ ID NO: 38. In certain aspects thepolypeptide subunit comprises the amino acid sequence SEQ ID NO: 56.

In certain aspects, the polypeptide subunit can comprise an antibody VLportion of a CD20 antigen binding domain as described above. In certainaspects the polypeptide subunit can comprise a human antibody lightchain constant region or fragment thereof fused to the C-terminal end ofa VL, where the VL comprises an LCDR1, LCDR2, and LCDR3, wherein theLCDR1 comprises the amino acid sequence of SEQ ID NO: 43, or SEQ ID NO:43 with one, two, three, four, or five single amino acid substitutions,e.g., one or two single amino acid substitutions; the LCDR2 comprisesthe amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 44 with one, two,three, four, or five single amino acid substitutions, e.g., one or twosingle amino acid substitutions; and the LCDR3 comprises the amino acidsequence of SEQ ID NO: 45 or SEQ ID NO: 45 with one, two, three, four,or five single amino acid substitutions, e.g., one or two single aminoacid substitutions; or the VL comprises an amino acid sequence at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 42. Incertain aspects the polypeptide subunit comprises the amino acidsequence SEQ ID NO: 58.

In certain aspects, the polypeptide subunit can comprise an antibody VHor an antibody VL portion of a CD20 antigen binding domain comprisingany one or more of the VH or VL amino acid sequences described aboveand/or in Table 5.

The disclosure further provides a composition comprising two or morepolynucleotides, where the two or more polynucleotides collectively canencode a dimeric, pentameric, or hexameric binding molecule as describedabove. In certain aspects the composition can include a polynucleotideencoding an IgA or IgM heavy chain or fragment thereof, e.g, a human IgAor IgM heavy chain as described above where the IgA or IgM heavy chaincomprises at least the VH of a CD20 antigen binding domain, and apolynucleotide encoding a light chain or fragment thereof, e.g., a humankappa or lambda light chain that comprises at least the VL of a CD20antigen binding domain. A polynucleotide composition as provided canfurther include a polynucleotide encoding a J-chain, e.g., a humanJ-chain, or a fragment thereof or a variant thereof. In certain aspectsthe polynucleotides making up a composition as provided herein can besituated on two or three separate vectors, e.g., expression vectors.Such vectors are provided by the disclosure. In certain aspects two ormore of the polynucleotides making up a composition as provided hereincan be situated on a single vector, e.g., an expression vector. Such avector is provided by the disclosure.

The disclosure further provides a host cell, e.g., a prokaryotic oreukaryotic host cell, comprising a polynucleotide or two or morepolynucleotides encoding a dimeric, pentameric, or hexameric CD20binding molecule as provided herein, or any subunit thereof, apolynucleotide composition as provided herein, or a vector or two,three, or more vectors that collectively encode a dimeric, pentameric,or hexameric CD20 binding molecule as provided herein, or any subunitthereof. In certain aspects a host cell provided by the disclosure canexpress a dimeric, pentameric, or hexameric CD20 binding molecule asprovided by this disclosure, or a subunit thereof.

In a related aspect, the disclosure provides a method of producing adimeric, pentameric, or hexameric CD20 binding molecule as provided bythis disclosure, where the method comprises culturing a host cell asdescribed above and recovering the binding molecule.

METHODS OF USE

This disclosure provides improved methods for directingcomplement-mediated, T-cell-mediated, or both complement-mediated andT-cell-mediated killing of cells that express CD20, e.g., B cells, e.g.,malignant or immortalized B cells, using a dimeric, pentameric, orhexameric IgA- or IgM-based CD20 binding molecule. The methods describedbelow can utilize binding molecules comprising CD20 antigen bindingdomains derived from any CD20 antibody, including without limitation1.5.3 as disclosed in U.S. Patent Publication No. 2007-0014720,rituximab, ofatumumab, veltuzumab, ocaratuzumab, obinutumumab, orvariants, derivatives, or analogs thereof, where the dimeric,pentameric, or hexameric CD20 binding molecule can provide improvedcomplement-mediated, T-cell-mediated, or both complement-mediated andT-cell-mediated killing of CD20-expressing cells as compared to anequivalent IgG antibody, fragment, variant, derivative, or analogthereof, comprising the same antigen binding domain. In certain aspectsthe IgA- or IgM-based CD20 binding molecule further comprises a J-chain,either a wild-type J-chain or a modified J-chain capable of binding tohuman CD3, e.g., V15J or J15V as provided herein. Based on thisdisclosure, construction of a dimeric, pentameric, or hexameric IgA- orIgM-based CD20 binding molecule comprising any CD20 antigen bindingdomain of interest is well within the capabilities of a person ofordinary skill in the art. The improved activity can, for example, allowa reduced dose to be used, or can result in more effective killing ofcells that are resistant to killing by the original antibody. By“resistant” is meant any degree of reduced activity of a CD20 antibody,e.g., rituximab, on the CD20-expressing cell. Use of an IgA-basedbinding molecule can allow, for example, greater tissue distribution fora binding molecule provided herein.

In certain aspects, this disclosure provides a method for directingimproved complement-mediated, T-cell-mediated, or bothcomplement-mediated and T-cell-mediated killing of a CD20-expressingcell, where the method includes contacting a CD20-expressing cell with adimeric, pentameric, or hexameric binding molecule as described herein,where the binding molecule can direct complement-mediated,T-cell-mediated, or both complement-mediated and T-cell-mediated killingof a CD-20-expressing cell, e.g., a CD20-expressing B cell, at higherpotency than an equivalent amount of a monospecific, bivalent IgG, e.g.,IgG1 antibody or fragment thereof that specifically binds to the sameCD20 epitope as the CD20 antigen binding domain, e.g., rituximab, whichcomprises a human IgG1 and a VH with the amino acid sequence SEQ ID NO:1 and a VL with the amino acid sequence SEQ ID NO: 5. The dimeric orpentameric binding molecule can further include a wild-type J-chain or amodified J-chain capable of binding to human CD3, e.g., V15J or J15V asprovided herein. In certain aspects a dimeric, pentameric, or hexamericbinding molecule as provided herein can direct complement-mediated,T-cell-mediated, or both complement-mediated and T-cell-mediated killingof a CD20-expressing cell, e.g., a CD20-expressing B cell at higherpotency than an equivalent amount of monospecific, bivalent CD20monoclonal antibody or fragment thereof, where the antibody is, orcomprises the same VH and VL regions as, e.g., ofatumumab, veltuzumab,ocaratuzumab, or obinutumumab.

This disclosure thus provides a method for directingcomplement-mediated, T-cell-mediated, or both complement-mediated andT-cell-mediated killing of a CD20-expressing cell, where the methodincludes: contacting a CD20-expressing cell with a dimeric, pentameric,or hexameric binding molecule comprising two, five, or six bivalentbinding units, respectively, wherein each binding unit comprises two IgAor IgM heavy chain constant regions or fragments thereof and two antigenbinding domains. Non-limiting examples of suitable binding moleculesinclude 1.5.3-based dimeric, pentameric, or hexameric binding moleculeprovided by this disclosure, or other binding molecules as describedelsewhere in this disclosure. The dimeric or pentameric binding moleculecan further include a wild-type J-chain or a modified J-chain capable ofbinding to human CD3, e.g., V15J or J15V as provided herein. Accordingto the method at least one antigen binding domain of the bindingmolecule is a CD20 antigen binding domain. Moreover, according to themethod the binding molecule can direct complement-mediated,T-cell-mediated, or both complement-mediated and T-cell-mediated killingof a CD-20-expressing cell at higher potency than an equivalent amountof a monospecific, bivalent IgG, e.g., IgG1 antibody or fragment thereofthat specifically binds to the same CD20 epitope as the CD20 antigenbinding domain, e.g., a bivalent IgG1 antibody comprising a CD20 antigenbinding domain similar to, e.g., identical to, a CD20 antigen bindingdomain of the dimeric, pentameric, or hexameric binding moleculeprovided by this disclosure.

For example, the disclosure provides a method for directingcomplement-mediated, T-cell-mediated, or both complement-mediated andT-cell-mediated killing of a CD20-expressing cell, where the methodincludes: contacting a CD20-expressing cell with a dimeric, pentameric,or hexameric binding molecule comprising at least one antigen bindingdomain related to 1.5.3, as described elsewhere herein, where thebinding molecule can direct complement-mediated, T-cell-mediated, orboth complement-mediated and T-cell-mediated killing of aCD-20-expressing cell at higher potency than an equivalent amount of amonospecific, bivalent IgG1 antibody or fragment thereof thatspecifically binds to the same CD20 epitope as 1.5.3-related CD20antigen binding domain. The dimeric or pentameric binding molecule canfurther include a wild-type J-chain or a modified J-chain capable ofbinding to human CD3, e.g., V15J or J15V as provided herein. In certainaspects the monospecific, bivalent IgG1 antibody is 1.5.3, and comprisesa VH having the amino acid sequence SEQ ID NO: 38 and a VL having theamino acid sequence SEQ ID NO: 42.

In certain aspects, e.g., where the binding molecule is a pentamericbinding molecule comprising five identical binding units each comprisingtwo identical binding domains with the VH and VL of 1.5.3, tested in aCDC assay using the CD20-expressing Raji cell line, the binding moleculecan direct complement-mediated killing with an IC₅₀ at least one-fold,at least two-fold, at least three-fold, at least four-fold, at leastfive-fold, at least ten-fold, at least 20-fold, at least thirty-fold, atleast forty-fold, at least 50-fold, at least 100-fold, at least150-fold, at least 200-fold or more lower than the IC₅₀ of an equivalentamount of the monospecific bivalent IgG1 antibody, e.g., 1.5.3, asmeasured, e.g., in μg/ml or in molar equivalents.

In certain aspects the CD-20-expressing cell is an immortalized cellline, e.g. a B cell leukemia or lymphoma cell line. The cell line canbe, without limitation, a Ramos cell line, a Raji cell line, a Daudicell line, a Namalwa cell line, a Granta cell line, a Z138 cell line, aDoHH2 cell line, or a DB cell line. Other cell lines that can be usefulin the methods provided herein can easily be identified by a person ofordinary skill in the art.

In certain aspects the cell line is a Granta cell line, and the dimeric,pentameric, or hexameric binding molecule can direct complement-mediatedkilling of the cell line at about six times the potency of rituximab asmeasured in μg/ml. In certain aspects the cell line is a Ramos cellline, and the dimeric, pentameric, or hexameric binding molecule candirect complement-mediated killing of the cell line at about 30-40 timesthe potency of rituximab as measured in molar equivalents.

In certain aspects the cell line is a Raji cell line or a Ramos cellline, and the dimeric, pentameric, or hexameric binding molecule candirect complement-mediated killing of the cell line at about three timesthe potency of rituximab.

In certain aspects the CD20-expressing cell is a malignant B cell in asubject, e.g., a human patient, with cancer. The cancer can be, forexample, a CD20-positive leukemia, lymphoma, or myeloma. In certainaspects the CD20-expressing cell line or malignant B cell is resistant,e.g., minimally responsive or non-responsive to killing by acommercially-available CD20 monoclonal antibody, e.g., theCD20-expressing cell line or malignant B cell in a subject with canceris minimally responsive or non-responsive to rituximab therapy.

In another aspect the disclosure provides a method for directingcomplement-mediated, T-cell-mediated, or both complement-mediated andT-cell-mediated killing of a CD20-expressing cell, where the methodincludes: contacting a CD20-expressing cell with a dimeric, pentameric,or hexameric binding molecule comprising at least one antigen bindingdomain related to the CD20 mAb rituximab, or a fragment, variant,derivative, or analog thereof. The dimeric or pentameric bindingmolecule can further include a wild-type J-chain or a modified J-chaincapable of binding to human CD3, e.g., V15J or J15V as provided herein.

In certain aspects the CD20 antigen binding domain of the dimeric,pentameric, or hexameric binding molecule comprises six immunoglobulincomplementarity determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,and LCDR3, wherein at least one, at least two, at least three, at leastfour, at least five, or at least six CDRs are related to thecorresponding CDRs of the CD20 mAb rituximab. The CD20 antigen bindingdomain can include an HCDR1 comprising the amino acid sequence of SEQ IDNO: 2 or SEQ ID NO: 2 with one, two, three, four, or five single aminoacid substitutions, e.g. one or two single amino acid substitutions. TheCD20 antigen binding domain can include an HCDR2 comprising the aminoacid sequence of SEQ ID NO: 3 or SEQ ID NO: 3 with one, two, three,four, or five single amino acid substitutions. For example, the HCDR2can comprise the amino acid sequence SEQ ID NO: 16. The CD20 antigenbinding domain can include an HCDR3 comprising the amino acid sequenceof SEQ ID NO: 4, or SEQ ID NO: 4 with one, two, three, four, or fivesingle amino acid substitutions, e.g., one, two, or three single aminoacid substitutions. For example, the HCDR3 can comprise the amino acidsequence SEQ ID NO: 17, SEQ ID NO: 31, or SEQ ID NO: 36. The CD20antigen binding domain can, in other aspects, include an HCDR3comprising the amino acid sequence SEQ ID NO: 10, or SEQ ID NO: 10 withone, two, three, four, or five single amino acid substitutions. The CD20antigen binding domain can, in other aspects, include an HCDR3comprising the amino acid sequence SEQ ID NO: 31, or SEQ ID NO: 31 withone, two, three, four, or five single amino acid substitutions. The CD20antigen binding domain can include an LCDR1 comprising the amino acidsequence of SEQ ID NO: 6, or SEQ ID NO: 6 with one, two, three, four, orfive single amino acid substitutions, e.g., one, two, or three singleamino acid substitutions. For example, the LCDR1 can comprise the aminoacid sequence SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO:27, or SEQ ID NO: 33. The CD20 antigen binding domain can include anLCDR2 comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 7with one, two, three, four, or five single amino acid substitutions,e.g., one or two single amino acid substitutions. For example, the LCDR2can comprise the amino acid sequence SEQ ID NO: 13 or SEQ ID NO: 20. TheCD20 antigen binding domain can include an LCDR3 comprising the aminoacid sequence of SEQ ID NO: 8 or SEQ ID NO: 8 with one, two, three,four, or five single amino acid substitutions, e.g., one or two singleamino acid substitutions. For example, the LCDR3 can comprise the aminoacid sequence SEQ ID NO: 14, SEQ ID NO: 21, or SEQ ID NO: 34.

In certain aspects the CD20 antigen binding domain of the dimeric,pentameric, or hexameric binding molecule comprises a VH and a VL,wherein the VH region, the VL region, or both the VH and the VL regionsare related to the corresponding VH and VL of rituximab. In certainaspects the CD20 antigen binding domain can comprise a VH amino acidsequence at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or 100% identical toSEQ ID NO: 1 and a VL amino acid sequence at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95% or 100% identical to SEQ ID NO: 5.

In certain aspects the VH and VL can be derived from the CD20 mAbdescribed in U.S. Pat. No. 7,679,900. For example, the CD20 antigenbinding domain can comprise a VH amino acid sequence at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identical to SEQ ID NO: 9 and a VL aminoacid sequence at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or 100% identical toSEQ ID NO: 11.

In certain aspects the VH and VL can be derived from the CD20 mAbdescribed in U.S. Pat. No. 8,153,125. For example, the CD20 antigenbinding domain can comprise a VH amino acid sequence at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identical to SEQ ID NO: 15 and a VLamino acid sequence at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to SEQ ID NO: 18.

In certain aspects the VH and VL can be derived from the CD20 mAbdescribed in U.S. Pat. No. 8,337,844. For example, the CD20 antigenbinding domain can comprise a VH amino acid sequence at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identical to SEQ ID NO: 22 or SEQ ID NO:23 and a VL amino acid sequence at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% or 100% identical to SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, orSEQ ID NO: 29.

In certain aspects the VH and VL can be derived from the CD20 mAbdescribed in U.S. Pat. No. 8,337,844. For example, the CD20 antigenbinding domain can comprise a VH amino acid sequence at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identical to SEQ ID NO: 30 and a VLamino acid sequence at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to SEQ ID NO: 32.

In certain aspects the VH and VL can be derived from the CD20 mAbdescribed in U.S. Pat. No. 7,151,164. For example, the CD20 antigenbinding domain can comprise a VH amino acid sequence at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or 100% identical to SEQ ID NO: 35 and a VLamino acid sequence at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%identical to SEQ ID NO: 37.

A dimeric, pentameric, or hexameric binding molecule for use in themethods provided herein, is a binding molecule with two, five, or six“binding units” as defined herein, that can specifically bind to CD20,e.g., human CD20. In certain aspects, a dimeric, pentameric, orhexameric binding molecule for use in the methods provided hereincomprises two, five, or six bivalent binding units, respectively, whereeach binding unit includes two IgA or IgM heavy chain constant regionsor fragments thereof. In certain aspects, the two IgA or IgM heavy chainconstant regions are human heavy chain constant regions.

Where the binding molecule for use in the methods provided herein ispentameric, the binding molecule can further comprise a J-chain, orfragment thereof, or variant thereof. The J-chain can be a wild-typeJ-chain or a modified J-chain capable of binding to human CD3, e.g.,V15J or J15V as provided herein.

An IgM heavy chain constant region of a binding molecule for use in themethods provided herein can include one or more of a Cμ1 domain, a Cμ2domain, a Cμ3 domain, and/or a Cμ4 domain, provided that the constantregion can serve a desired function in the binding molecule, e.g.,associate with second IgM constant region to form an antigen bindingdomain, or associate with other binding units to form a hexamer or apentamer. In certain aspects the two IgM heavy chain constant regions orfragments thereof within an individual binding unit each comprise a Cμ3domain or fragment thereof, a Cμ4 domain or fragment thereof, atailpiece (TP) or fragment thereof, or any combination of a Cμ3 domain aCμ domain, and a TP or fragment thereof. In certain aspects the two IgMheavy chain constant regions or fragments thereof within an individualbinding unit each further comprise a Cμ2 domain or fragment thereof, aCμ1 domain or fragment thereof, or a Cμ1 domain or fragment thereof anda Cμ2 domain or fragment thereof.

While a variety of different dimeric, pentameric, or hexameric bindingmolecules for use in the methods provided herein can be contemplated bya person of ordinary skill in the art based on this disclosure, and assuch are included in this disclosure, in certain aspects, a bindingmolecule for use in the methods provided herein is provided in whicheach binding unit comprises two IgA or IgM heavy chains each comprisinga VH situated amino terminal to the IgA or IgM constant region orfragment thereof, and two immunoglobulin light chains each comprising aVL situated amino terminal to an immunoglobulin light chain constantregion.

Moreover, in certain aspects, at least one binding unit of the bindingmolecule for use in the methods provided herein, or at least two, atleast three, at least four, at least five, or at least six binding unitsof the binding molecule for use in the methods provided herein,comprises or comprise two of the CD20 antigen binding domains asdescribed above. In certain aspects the two CD20 antigen binding domainsin the one, two, three, four, five, or six binding unit(s) of thebinding molecule for use in the methods provided herein can be differentfrom each other, or they can be identical.

In certain aspects, the two IgM heavy chains within the one, two, three,four, five, or six binding unit(s) of the binding molecule for use inthe methods provided herein are identical. In certain aspects, twoidentical IgM heavy chains within at least one binding unit, or withinat least two, at least three, at least four, at least five, or at leastsix binding units of the binding molecule for use in the methodsprovided herein comprise the amino acid sequence SEQ ID NO: 52.

In certain aspects, the two light chains within the one, two, three,four, five, or six binding unit(s) of the binding molecule for use inthe methods provided herein are identical. In certain aspects, twoidentical light chains within at least one binding unit, or within atleast two, at least three, at least four, at least five, or at least sixbinding units of the binding molecule for use in the methods providedherein are kappa light chains, e.g., human kappa light chains, or lambdalight chains, e.g., human lambda light chains. In certain aspects, twoidentical light chains within at least one binding unit, or within atleast two, at least three, at least four, at least five, or at least sixbinding units of the binding molecule for use in the methods providedherein each comprise the amino acid sequence SEQ ID NO: 54.

In certain aspects at least one, at least two, at least three, at leastfour, at least five, or at least six binding units of a pentameric orhexameric binding molecule for use in the methods provided hereincomprises or each comprise two identical IgM heavy chains eachcomprising the amino acid sequence SEQ ID NO: 52, and two identicallight chains each comprising the amino acid sequence SEQ ID NO: 54.According to this aspect, the CD20 antigen binding domains in the one,two, three, four, five, or six binding unit(s) of the binding moleculecan be identical. Further according to this aspect, a dimeric,pentameric, or hexameric binding molecule for use in the methodsprovided herein can comprise at least one, at least two, at least three,at least four, at least five, at least six, at least seven, at leasteight, at least nine, at least ten, at least eleven, or at least twelvecopies of a CD20 antigen binding domain as described above. In certainaspects at least two, at least three, at least four, at least five, orat least six of the binding units can be identical and, in certainaspects the binding units can comprise identical antigen bindingdomains, e.g., at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, or at least twelve CD20 antigen bindingdomains can be identical. In certain aspects the identical CD20 antigenbinding domain can comprise a VH with the amino acid sequence SEQ ID NO:1 and a VL with the amino acid sequence SEQ ID NO: 5.

Dimeric, pentameric, or hexameric CD20 binding molecules for use in themethods provided herein can possess advantageous structural orfunctional properties compared to other binding molecules. For example,a dimeric, pentameric, or hexameric CD20 binding molecule for use in themethods provided herein can possess improved activity in a biologicalassay, either in vitro or in vivo, than a corresponding bindingmolecule, e.g., rituximab or a variant, analog, or derivative thereof.Biological assays include, but are not limited to, complement-dependentcellular cytotoxicity (CDC) and/or antibody dependent cellularcytotoxicity (ADCC).

Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering a dimeric, pentameric, orhexameric CD20 binding molecule as provided herein to a subject in needthereof are well known to or are readily determined by those skilled inthe art in view of this disclosure. The route of administration of aCD20 binding molecule can be, for example, oral, parenteral, byinhalation or topical. The term parenteral as used herein includes,e.g., intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal, or vaginal administration. While these forms ofadministration are contemplated as suitable forms, another example of aform for administration would be a solution for injection, in particularfor intravenous or intraarterial injection or drip. A suitablepharmaceutical composition can comprise a buffer (e.g. acetate,phosphate or citrate buffer), a surfactant (e.g. polysorbate),optionally a stabilizer agent (e.g. human albumin).

As discussed herein, a dimeric, pentameric, or hexameric CD20 bindingmolecule as provided herein can be administered in a pharmaceuticallyeffective amount for the in vivo treatment of diseases or disorders inwhich it's desirable to deplete B cells. In this regard, it will beappreciated that the disclosed binding molecules can be formulated so asto facilitate administration and promote stability of the active agent.Pharmaceutical compositions accordingly can comprise a pharmaceuticallyacceptable, non-toxic, sterile carrier such as physiological saline,non-toxic buffers, preservatives and the like. A pharmaceuticallyeffective amount of a dimeric, pentameric, or hexameric CD20 bindingmolecule as provided herein means an amount sufficient to achieveeffective binding to a target and to achieve a therapeutic benefit.Suitable formulations are described in Remington's PharmaceuticalSciences (Mack Publishing Co.) 16th ed. (1980).

Certain pharmaceutical compositions provided herein can be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also can be administered by nasal aerosol or inhalation.Such compositions can be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of a dimeric, pentameric, or hexameric CD20 binding moleculethat can be combined with carrier materials to produce a single dosageform will vary depending, e.g., upon the subject treated and theparticular mode of administration. The composition can be administeredas a single dose, multiple doses or over an established period of timein an infusion. Dosage regimens also can be adjusted to provide theoptimum desired response (e.g., a therapeutic or prophylactic response).

In keeping with the scope of the present disclosure, a dimeric,pentameric, or hexameric CD20 binding molecule as provided herein can beadministered to a subject in need of therapy in an amount sufficient toproduce a therapeutic effect. A dimeric, pentameric, or hexameric CD20binding molecule as provided herein can be administered to the subjectin a conventional dosage form prepared by combining the antibody orantigen-binding fragment, variant, or derivative thereof of thedisclosure with a conventional pharmaceutically acceptable carrier ordiluent according to known techniques. The form and character of thepharmaceutically acceptable carrier or diluent can be dictated by theamount of active ingredient with which it is to be combined, the routeof administration and other well-known variables.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of a dimeric, pentameric, or hexameric CD20 bindingmolecule, that when administered brings about a positive therapeuticresponse with respect to treatment of a patient with a disease orcondition to be treated.

Therapeutically effective doses of the compositions disclosed herein,for treatment of diseases or disorders in which B cell depletion isdesired, can vary depending upon many different factors, including meansof administration, target site, physiological state of the patient,whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic. Incertain aspects, the subject or patient is a human, but non-humanmammals including transgenic mammals can also be treated. Treatmentdosages can be titrated using routine methods known to those of skill inthe art to optimize safety and efficacy.

The amount of a dimeric, pentameric, or hexameric CD20 binding moleculeto be administered is readily determined by one of ordinary skill in theart without undue experimentation given this disclosure. Factorsinfluencing the mode of administration and the respective amount of adimeric, pentameric, or hexameric CD20 binding molecule include, but arenot limited to, the severity of the disease, the history of the disease,and the age, height, weight, health, and physical condition of theindividual undergoing therapy. Similarly, the amount of a dimeric,pentameric, or hexameric CD20 binding molecule to be administered willbe dependent upon the mode of administration and whether the subjectwill undergo a single dose or multiple doses of this agent.

This disclosure also provides for the use of a dimeric, pentameric, orhexameric CD20 binding molecule in the manufacture of a medicament fortreating, preventing, or managing a disease or disorder in which B celldepletion is desirable, e.g., B cell lymphoma, leukemia, or myeloma.

This disclosure employs, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Sambrook et al., ed. (1989) MolecularCloning A Laboratory Manual (2nd ed.; Cold Spring Harbor LaboratoryPress); Sambrook et al., ed. (1992) Molecular Cloning: A LaboratoryManual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985)DNA Cloning, Volumes I and II; Gait, ed. (1984) OligonucleotideSynthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins,eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984)Transcription And Translation; Freshney (1987) Culture Of Animal Cells(Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986);Perbal (1984) A Practical Guide To Molecular Cloning; the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Caloseds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold SpringHarbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In CellAnd Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (RL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described can be followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freeman & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlag); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1: Generation of DNA Constructs for Expression ofCD20-hIgM

Plasmid constructs that can express pentameric or hexameric IgM bindingmolecules that can specifically bind to CD20 were produced by thefollowing method.

DNA fragments encoding the VH and VL regions of rituximab (SEQ ID NOs 1and 5, respectively) or 1.5.3 (SEQ ID NOs 38 and 42, respectively) weresynthesized by a commercial vendor (Genescript), with an EcoRVrestriction site on the ′5 end and an XbaI restriction site on the 3′end for subcloning into heavy chain and light chain expression vectors.The synthesized DNA constructs were re-suspended in Tris-EDTA buffer at1 μg/ml. DNA samples (1 μg) were digested with EcoRV and XbaI, and thesynthesized VH and VL were separated from the carrier plasmid DNA byelectrophoresis. The digested DNA was ligated to pre-digested plasmidDNA (pFUSEss-CHIg-hM*03 for μ chain, pFUSE2ss-CLIg-hk for kappa chain,available from Invivogen) by standard molecular biology techniques. Theligated DNAs were transformed into competent bacteria and plated on LBplates with multiple selective antibiotics. Several bacterial colonieswere picked, and DNA preparations were made by standard molecularbiology techniques. The constructs encoding the heavy chain and lightchains were verified by sequencing. The DNA and amino acid sequences ofthe rituximab IgM heavy chain are presented as SEQ ID NO: 51 and SEQ IDNO: 52, respectively, and the DNA and amino acid sequences of therituximab light chain are presented as SEQ ID NO: 53 and SEQ ID NO: 54,respectively. The DNA and amino acid sequences of 1.5.3 IgM heavy chainare presented as SEQ ID NO: 55 and SEQ ID NO: 56, respectively, and theDNA and amino acid sequences of 1.5.3 light chain are presented as SEQID NO: 57 and SEQ ID NO: 58, respectively. The amino acid sequence ofthe human J-chain is presented as SEQ ID NO: 49.

The plasmid constructs encoding the IgM heavy chains and light chains,or the heavy chains, light chains, and J-chain were cotransfected intoCHO cells, and cells that express the CD20 IgM antibody, either with orwithout J-chain, were selected, all according to standard methods.

Antibodies present in the cell supernatants were recovered using CaptureSelect IgM (Catalog 2890.05, BAC, Thermo Fisher) according to themanufacturer's protocol. Antibodies were evaluated on SDS PAGE undernon-reducing conditions to show assembly of pentamers and hexamers.NuPage LDS Sample Buffer (Life Technologies) was added to samples beforeloading onto a NativePage Novex 3-12% bis-Tris Gel (Life TechnologiesCatalog #BN1003). Novex Tris-Acetate SDS Running Buffer (LifeTechnologies Catalog #LA0041) was used for gel electrophoresis. The gelwas run until the dye front reached the bottom of the gel. Afterelectrophoresis, the gel was stained with Colloidal Blue Stain (LifeTechnologies Catalog #LC6025). The results are shown in FIG. 4. Undernon-reducing conditions, the pentameric 1.5.3 IgM (five H2L2 IgM unitsplus J-chain, FIG. 4, second panel, lane 3) and the pentameric rituximabIgM (first panel, lane 3) produced a protein band of approximately1,000,000 molecular weight, and hexameric 1.5.3 IgM (six H2L2 IgM units,FIG. 4, second panel, lane 2) produced a protein band of approximately1,180,000.

Example 2: Binding and Activity of CD20-hIgM

Detection of Binding via CD20 ELISA Assay

96-well white polystyrene ELISA plates (Pierce 15042) were coated with100 μL per well of 10 μg/mL or 0.3 μg/mL human CD20 with N-Fc fusion(AcroBiosystems, CDO-H526a) overnight at 4° C. Plates were then washedwith 0.05% PBS-Tween and blocked with 2% BSA-PBS. After blocking, 100 μLof serial dilutions of 1.5.3 IgM, 1.5.3 IgG, standards, and controlswere added to the wells and incubated at room temperature for 2 hours.The plates were then washed and incubated with HRP conjugated mouseanti-human kappa (Southern Biotech, 9230-05. 1:6000 diluted in 2%BSA-PBS) for 30 min. After 10 final washes using 0.05% PBS-Tween, theplates were read out using SuperSignal chemiluminescent substrate(ThermoFisher, 37070). Luminescent data were collected on an EnVisionplate reader (Perkin-Elmer) and analyzed with GraphPad Prism using a4-parameter logistic model.

The results are shown in FIG. 5A and FIG. 5B, comparing IgM vs. IgG bymolar concentrations. The anti-CD20 IgM antibody exhibited moreeffective binding, at both CD20 antigen densities, and especially at thelow CD20 antigen concentration (FIG. 5B).

Complement Dependent Cytotoxicity—Colorimetric Assay

Granta (DSMZ cat. #ACC 342), Raji (ATCC cat. #CCL-86), Ramos (ATCC cat.#CRL-1596), Nalm-6 (DSMZ cat. #ACC 128), and Namalwa (ATCC cat.#CRL-1432) cell lines were from ATCC and DSMZ. 50,000 cells of each cellline were seeded in a 96-well plate. Cells were treated with seriallydiluted commercially-available anti-human CD20 IgM (Invivogen cat.#hcd20-mab5) or anti-human CD20 IgG1 (Invivogen cat. #hcd20-mabl). Humanserum complement (Quidel cat. #A113) was added to each well at a finalconcentration of 10%. The reaction mixtures were incubated at 37° C. for1 hr. Cell Counting Kit-SK reagent (CCK-SK) (Dojindo cat. #CK04-13) wasadded at 1/10 the total reaction volume and plate was incubated for anadditional 3 hours at 37° C. Absorbance at 450 nm was measured on aspectrophotometer.

The results are shown in FIG. 6A-E. The IgM CD20 antibody was 6 timesmore potent at cell killing than IgG in Granta cells (FIG. 6A), threetime more potent in Raji cells (FIG. 6B), and three times more potent inRamos cells (FIG. 6C). Neither antibody was effective in killing Nalm-6cells (FIG. 6D) or Namalwa cells (FIG. 6E), which express no, or lowlevels of CD20, respectively.

Complement Dependent Cytotoxicity—Luminescent Assay

(a) The CD20-expressing Raji cell line (ATCC cat. #CCL-86) was used.50,000 cells were seeded in a 96-well plate. Cells were treated with thefollowing serially diluted antibodies: rituximab (IgG1),rituximab-derived anti-human CD20 IgM+J as produced in Example 1, or1.5.3 anti-human CD20 IgM+J, produced as described in Example 1. Humanserum complement (Quidel cat. #A113) was added to each well at a finalconcentration of 10%. The reaction mixtures were incubated at 37° C. for4 hours. Cell Titer Glo reagent (Promega cat. #G7572) was added at avolume equal to the volume of culture medium present in each well. Theplate was shaken for 2 minutes, incubated for 10 minutes at roomtemperature, and luminescence was measured on a luminometer.

The results are shown in FIG. 7 and Table 2. Anti-CD20 as an IgG(rituximab) achieved approximately half-maximal Raji cell killing withcomplement, whereas the IgM isotype (rituximab) achieved nearly maximalcomplement dependent cytotoxicity. Both IgM antibodies were more potentat complement dependent cytotoxicity than rituximab, and in thisexperiment, the 1.5.3-like IgM antibody was more potent than theanti-human IgM CD20 antibody carrying the rituximab VH and VL. The1.5.3-like IgM antibody exhibited four-fold increased potency comparedto that of the type 1 anti-CD20 (rituximab as IgM).

TABLE 2 CDC (IC₅₀) on Raji cells (μg/ml) IC₅₀ (μg/ml) Anti-CD20 IgG1(rituximab) >50 Anti-CD20 IgM 2.0 1.5.3 IgM + J 0.5

(b) The CDC assay as described above was repeated using theCD20-expressing Ramos cell line with the following serially dilutedantibodies: rituximab (IgG), rituximab-derived anti-human CD20 IgM+J asproduced in Example 1, 1.5.3 (IgG1), 1.5.3 anti-CD20 IgM, or 1.5.3anti-CD20 IgM+J, produced as described in Example 1.

The results are shown in FIG. 8A (rituximab and rituximab-like IgM) andFIG. 8B (1.5.3, 1.5.3 IgM+J, and huMAb-like IgM. In this experiment, theIgG and IgM versions were compared on a molar equivalent basis. TheIC₅₀s for the IgM versions were all about 30 to 40 times more effectiveat complement dependent cytotoxicity than the IgG versions.

(c) Next, the antibodies were then compared for CDC activity ondifferent cell lines with decreasing CD20 expression levels. The assaywas carried out with the following serially diluted antibodies:rituximab (IgG1), rituximab-derived anti-human CD20 IgM+J as produced inExample 1, 1.5.3 (IgG1), and 1.5.3 anti-CD20 IgM+J, produced asdescribed in Example 1. The cells used were DoHH2 cells (DSMZ No. ACC47), and Z138 cells (ATCC CRL-3001) were used in this assay. Z138 cellsexhibit lower expression levels of CD20 than DoHH2 cells, as shown Table4 below. The target cells were washed and resuspended in CDC assaymedium (RPMI 1640, 10% heat-inactivated FBS) at a density of 1.0×10⁶cells/mL and 10 μL/well was added to a Nunc 384-well tissueculture-treated white polystyrene plate. Serial 3-fold dilutions of testantibodies were prepared in assay medium, 10 μL/well was added to theassay plate, and the plate was incubated for 2 hr at 37° C. in a 5% CO₂incubator to allow opsonization to occur. Normal human serum complement(Quidel) was frozen in aliquots, thawed once for use, diluted to 30% inassay medium, and 10 μL/well was added to the assay plate. The plate wasincubated for 4 hr at 37° C. in a 5% CO₂ incubator. Cell Titer-Gloreagent (Promega) was thawed for use and 15 μL/well was added to theassay plate. The plate was gently mixed for 2 min on a plate shaker tolyse the cells and then for another 10 min at room temperature beforemeasuring luminescence on an EnVision plate reader (Perkin-Elmer). Aftersubtracting background signal, percent viability was plotted againstantibody concentration and EC50 values were determined using GraphPadPrism.

The results are shown in FIG. 9 and in Table 3. On both cell lines, theIgM versions of the antibodies exhibited greater CDC killing than theIgG versions. On the lower CD20-expressing Z-138 cells, the CDC activityof the IgM versions were more than 100-fold improved relative to the IgGversions.

TABLE 3 CDC Activity Depends on CD20 Antigen Expression Level EC₅₀ (nM)DOHH-2 Z-138 Anti-CD20 IgG1 (rituximab) 22 >100 1.5.3 IgG 4.6 >100Anti-CD20 IgM + J 0.09 0.41 1.5.3 IgM + J 0.14 1.9

Example 3: Preparation of a Bispecific Anti-CD20 IgM Comprising aModified J-Chain Binding CD3

Rituxan-like anti-human CD20 IgM and 1.5.3 anti-CD20 IgM were producedas described in Example 1.

Two different J-chain variants were constructed with distinct fusionsites incorporating variable regions from the anti-CD3 antibodyvisilizumab (Nuvion). Shown below are the sequences for two J-chainswith the scFv corresponding to visilizumab (V) (VH-(GGGGS)₃-VL,double-underlined) fused to the J-chain (italics) through a (GGGGS)₃linker (SEQ ID NO: 67, underlined) containing 15 amino acids in twodifferent orientations—V15J and J15V. Each sequence contains anN-terminal signal peptide that is shown without underlining or italics.In certain aspects, other signal peptide sequences can be substitutedfor the signal peptides shown here.

SEQ ID NO: 63: precursor modified J-chain sequence for V15J(DNA Sequence: SEQ ID NO: 68):MGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK GGGGSG GGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDSEQ ID NO: 64: mature modified J-chain sequence for V15J:QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGS QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKNIVETALTPDACYPDSEQ ID NO: 65: precursor modified J-chain sequence for J15V(DNA sequence: SEQ ID NO: 69):MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETK MVETALTPDACYPDGGGGSGGGGSGGGGS QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTK LEIKSEQ ID NO: 66: mature modified J-chain sequence for J15V:QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPTRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD GGGGSGGGG SGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK

The mature constructs each have a molecular weight of about 45 kD andcan bind to soluble epsilon chain of CD3 (Sino Biological), or T-cells(data not shown).

The DNA constructs corresponding to the anti-CD20 heavy and light chainsas well as those corresponding to either the wild-type (wt) J-chain,V15J or J15V J-chain sequences were cotransfected into mammalian cells,e.g., HEK293 or CHO cells, and proteins were expressed and purifiedaccording to standard methods. See, e.g., PCT Publication No. WO2015/153912, which is incorporated herein by reference in its entirety.The J-chains fused to the anti-CD3 scFv with the 15 amino acid linkerwere able to incorporate with the anti-CD20 heavy and light chains toproduce a pentameric form of bi-specific IgM.

Agarose-Acrylamide Hybrid Gel.

IgM Constructs were separated by non-reducing SDS-PAGE adapted from apreviously described method (Chugai Seiyaki Kabushiki Kaisha, 2010, Pub.No.: US 2010/0172899 A1). Briefly, the hybrid gel was mixed with 40%Acrylamide/Bis-Acrylamide, 37.5:1 (Sigma-Aldrich) and Ultrapure Agarose(Invitrogen) to final concentrations of 3.6% and 0.5%, respectively, in0.375 M Tris Buffer, pH 8.8 and 15% glycerol. The resulting mixture washeated to 50° C. and polymerization was initiated with the addition of0.08% TEMED and 0.08% of ammonium persulfate. The resulting solution waspoured between two plates and the acrylamide was allowed to polymerizeat 37° C. for 1 hour and then left at room temperature for 30 min toensure complete polymerization. Protein samples were loaded into theresulting hybrid gel and the gel was run in Tris-Acetate SDS RunningBuffer (Novex) for 800 Vh. The gel was then fixed in 40% methanol, 10%acetic for 10 minutes, stained using a Colloidal Blue Staining Kit(Novex) for at least 3 hours and subsequently de-stained in water.

Non-Reducing SDS-Native-PAGE.

Protein samples were loaded into a NativePAGE 3-12% Bis-Tris gel(Novex). Tris-Acetate SDS Running Buffer (Novex) was added and the gelwas run at 40V for 15 min and then at 90V for 2 hours. The gel was thenfixed in 40% methanol, 10% acetic acid for 10 minutes, stained using aColloidal Blue Staining Kit (Novex) for at least 3 hours andsubsequently de-stained in water.

J-Chain Western Blot.

An acrylamide gel run under reducing conditions was washed in a 20%ethanol solution for 10 minutes and then the protein was transferred toan iBlot PVDF membrane (Invitrogen) using the iBlot Dry Blotting System(Invitrogen) at 20V for 10 minutes. After transfer the PVDF membrane wasblocked using 2% bovine serum albumin, 0.05% Tween 20 for at least 12hours. A 1/500 dilution of Pierce J-chain antibody (ThermoFisher) wasadded to the membrane, incubated for 1 hour, and then a 1/5000 dilutionof peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch)was added and allowed to incubate in darkness for 30 minutes. Finally,Super Signal West Pico Chemiluminescent Substrate (ThermoFisher) wasadded to the blot and the resulting signal was visualized using theChemiDoc-It HR410 Imaging System (UVP) or by exposing the blot to X-rayfilm.

As shown in FIG. 10A and FIG. 10B, the 1.5.3 IgM+wt J and 1.5.3 IgM+V15Jproteins are visible as faster migrating pentamers distinguishable on ahybrid gel from the slower migrating hexamer form not containing aJ-chain. Integration of the J-chain is confirmed on the reducing gel(FIG. 10C) as well as western blot with antibodies to the J-chain (FIG.10D).

Example 4: T-Cell Activation Assay

To demonstrate that a bispecific anti-CD20/anti-CD3 antibody couldactivate T-cells upon binding to the CD20 target the following assay wasperformed. Engineered Jurkat T-cells (Promega CS176403) and RPMI8226cells (ATCC CCL-155) were cultured in RPMI (Invitrogen) supplementedwith 10% Fetal Bovine Serum (Invitrogen). Serial dilutions of purified1.5.3 IgM+V15J, Blinatumomab (bispecific CD19 x CD3), and monospecific1.5.3 IgM were incubated with 7500 RPMI8226 cells in 20 μL in a white384 well assay plate for 2 h at 37° C. with 5% CO₂. The engineeredJurkat cells (25000) were added to mixture to final volume of 40 μL. Themixture was incubated for 5 h at 37° C. with 5% CO₂. The cell mixtureswere then mixed with 20 μL lysis buffer containing luciferin (Promega,Cell Titer Glo) to measure luciferase reporter activity. Light outputwas measured by EnVision plate reader. EC50 was determined by 4parameter curve fit using Prism software.

The results are shown in FIG. 11. T-cell activation with the 1.5.3-V15Jantibody was greater than that seen with Blinatumomab on the RPMI8226cell line. The maximal level of T-cell activation showed goodcorrelation with the level of CD20 expression on cell surface as shownin FIG. 12 using a series of tumor cell lines each expressing adifferent level of CD20 antigen.

Example 5: T-cell Directed B-Cell Killing—LDH Release Assay

In order to demonstrate that bispecific CD20 x CD3 IgM binding moleculescan kill target cells in the presence of CD8+ T-cell acute lymphoblasticleukemia (TALL) cells, we performed co-culture experiments. 6×10³cancerous B cells were co-cultured with 3×10⁴ TALL cells (ATCCCRL-11386) in the presence of different concentrations of test compounds(Rituxan IgM+V15J and 1.5.3 IgM+V15J) in 45 μL total volume of RPMI 1640media supplemented with 10% heat-inactivated FBS per well on a 384-wellblack tissue culture plate. After 24 hours of incubation at 37° C. in a5% CO₂ incubator, 15 μL of CytoTox-ONE substrate reagent (Promega,G7891) was added to each well to measure the level of LDH released fromdead cells. The plates were shaken briefly to mix the reagents, and thenincubated at room temperature for 90 min before measuring fluorescencesignal (485 nm for excitation and 615 nm for emission) on an EnVisionplate reader (Perkin-Elmer). The data was then analyzed with GraphPadPrism to determine the EC₅₀. As shown Table 4, the EC₅₀ of cell killingcorrelated with the expression level of CD20 antigen on cell surfaceusing both 1.5.3 IgM V15J and Rituximab IgM V15J with EC50 on DB cellline as low as 0.4 ng/mL (0.4 pM).

TABLE 4 T-cell Directed B-Cell Killing EC₅₀ (ng/mL) Cell lines MFIRituxan IgM + V15J IgM 1.5.3 + V15J DB 3400 0.5 0.4 DOHH2 1300 13 12Z-138 800 30 33

Example 6: In Vitro Cytotoxicity Assay Using KILR™ Detection Kit

In order to examine the ability of 1.5.3 IgM+V15J to kill CD20+ tumorcells in whole blood (i.e., with the inclusion of T-cells andcomplement), the KILR™ in vitro cytotoxicity detection kit was used. TheKILR™ ARH-77 cell line (CD20+) was purchased from DiscoverX(97-1001C017) as target cells. 5-10×10³ KILR™ ARH-77 cells wereco-cultured with either human CD8+ T-cells (Precision for Medicine),PBMC (AllCells or Precision for Medicine), or Hirudin anti-coagulatedhuman blood (AllCells) in the presence of different concentrations oftest compounds (1.5.3 IgM+V15J, 1.5.3 IgM+wtJ, 1.5.3 IgG, Rituximab IgGand blinatumomab) in 200 μL total volume of RPMI 1640 media supplementedwith 10% heat-inactivated FBS (when human blood was used for co-culture,less media were used because the whole blood took up 20-50% of thevolume) on a 96-well U-bottom non-tissue culture-treated polystyreneplate (Corning Falcon). After 4-48 hours of incubation at 37° C. in a 5%CO₂ incubator, the plates were centrifuged at 27×g for 5 min. 50 μL ofthe supernatants were transferred to a 96-well flat-bottom whitepolystyrene plate (Greiner Bio-One) to be mixed with 25 μL of KILRdetection working solution (KILR™ detection kit: DiscoverX 97-0001M) fora 1-hour incubation at room temperature before measuring luminescence onan EnVision plate reader (Perkin-Elmer). The target cells were lysedwith the lysis buffer supplied with the KILR™ detection kit to establishtotal lysis control. Percent killing was plotted against antibodyconcentration and EC50 values were determined using GraphPad Prism.

The results in the presence of normal human complement in whole blood(with hirudin) are shown in FIG. 13. The 1.5.3 IgM+wtJ (diamonds) andIgM+V15J (squares) antibodies both showed very potent killing at fourhours. Rituxan IgG (open circles) and Blinatumomab (triangles) were atleast 30 times less potent.

Example 7: In Vivo Efficacy Study Using Humanized NOD/SCID GammaKnock-Out (NSG) Mouse Models

CD34+ humanized NSG mouse studies were performed by In-VivoTechnologies, Inc. These mice are surrogates for human immuno-oncologystudies, in that they possess develop multi-lineage human immune cells.The mice were purchased from the Jackson Laboratory, and dosed with testarticles through tail vein injection. In addition to a vehicle control,the test articles included 1.5.3 IgM+V15J at 3 g, 1 g, and 0.3 g permouse and rituximab at 1 g and 0.3 g per mouse. Blood samples werecollected at 6 h, 24 h, and 10 days post dose through facial vein. Forthe PBMC humanized NSG mouse studies, frozen PBMCs from AllCells weresent to the Jackson Laboratory for injection. Each NSG mouse wasinjected with 10 million PBMCs after 100 cGy whole body irradiation.Blood samples were collected before and after tail vein dosing of testarticles (as above) via retro-orbital bleeding at designated timepoints. Blood samples from both the CD34+ and PBMC mouse studies weresent back to IGM Biosciences Inc. for lymphocyte analysis. Blood sampleswere stained for human CD56, CD3, CD19 and CD45 markers to identifydifferent population of human lymphocytes. CountBright Absolute CountingBeads (LifeTechnologies, C36950) were used to quantify the absolutenumber of lymphocytes in the blood samples. The lymphocyte levels wereplotted and analyzed using GraphPad Prism.

Results at 6 hours post-dose for 1.5.3 IgM with wild-type J and 1.5.3IgM with V15J, normalized to pre-dose B cell levels, are shown in FIG.14A and FIG. 14B, respectively. The bispecific 1.5.3 IgM x V15J antibodyshowed potent T-cell dependent B-cell killing in the engrafted NSG micewith as little as 3 g per mouse.

A comparison of the results over the full assay period between 1.5.3 IgMx V15J and rituximab are shown in FIG. 15.

TABLE 5 Sequences in the Disclosure SEQ ID NO Short Name Sequence  1Rituximab VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTT VTVSA  2Rituximab SYNMH HCDR1  3 Rituximab AIYPGNGDTSYNQKFKG HCDR2  4 RituximabSTYYGGDWYFNV HCDR3  5 Rituximab VLQIVLSQSPAILSASPGEKVTMTCRASSSVSYTHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKR  6 RituximabRASSSVSYIH LCDR1  7 Rituximab ATSNLAS LCDR2  8 Rituximab QQWTSNPPT LCDR3 9 900 VH EVQLVESGGG LVQPGGSLRL SCAASGYTFT SYNMHWVRQA PGKGLEWVGAIYPGNGDTSY NQKFKGRFTI SVDKSKNTLY LQMNSLRAED TAVYYCARVVYYSNSYWYFD VWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCLVKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT 10 900HCDR3VVYYSNSYWYFDV 11 900 VLDIQMTQSPSS LSASVGDRVT ITCRASSSVS YMHWYQQKPG KAPKPLIYAPSNLASGVPSR FSGSGSGTDF TLTISSLQPE DFATYYCQQW SFNPPTFGQGTKVEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVDNALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL 12 900LCDR1RASSSVSYMH 13 900LCDR2 APSNLAS 14 900LCDR3 QQWSFNPPT 15 125 VHEVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDTSYNQKSKLQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYVGGDWQFDVWGKGTT VTVSS 16125HCDR2 aiypltgdtsynqkskl 17 125HCDR3 styvggdwqfdv 18 125 VLEIVLTQSPGTLSLSPGERATLSCRASSSVPYIHWYQQKPGQAPRLLIYATSALASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSNPPTFGQGTKLEIK 19 125LCDR1 RASSSVPYIH20 125LCDR2 ATSALAS 21 125LCDR3 QQWLSNPPT 22 844 VH #2QVQLQQPGAELKKPGASVKVSCKASGYTFTSYNMHWVKQTPGRGLEWTGAIYPGNGDTSYNQKFKGKTTLTADKSSSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGAGTT VTVSA 23844 VH #3 QVQLQQPGAELKKPGASVKVSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKTTLTADKSSSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGAGTT VTVSA 24844 VL #5 QIVLSQSPAIITASPGEKVTMTCRASTSASYTHWFQQKPTSSPKPWIYATSNLASGVPSRFSGSGSGTTYSMTISSLEAEDAATYYCQQWTSNPPTFGGGTKLEIK 25 844 VL #5 RASTSASYIHLCDR1 26 844 VL #6QIVLSQSPAIITASPGEKVTMTCRASTSVSYIHWFQQKPTSSPKPWIYATSNLASGVPSRFSGSGSGTTYSMTISSLEAEDAATYYCQQWTSNPPTFGGGTKLEIK 27 844 VL  RASTSVSYIHLCDR1 28 844 VL #7QIVLSQSPAIITASPGEKVTMTCRASTSVSYIHWFQQKPGSSPKPWIYATSNLASGVPSRFSGSGSGTTYSMTISSLEAEDAATYYCQQWTSNPPTFGGGTKLEIK 27 null 29 844 VL #8QIVLSQSPAIITASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPSRFSGSGSGTTYSMTISSLEAEDAATYYCQQWTSNPPTFGGGTKLEIK 30 844 VH #10EVQLQQSGAELKKPGASVKVSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKTTLTADKSSSTAYMELSSLRSEDTAVYYCARSNYYGSSYWFFDVWGTGT TVTVSS 31844 VH #10 SNYYGSSYWFFDV HCDR3 32 844 VL #12DIVLTQSPAIITASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPSRFSGSGSGTTYSMTISSLEAEDAATYYCQQWSFNPPTFGGGTKLEIK 33 844 VL #12RASSSVNYMD LCDR1 34 844 VL #12 QQWSFNPPT LCDR3 35 164 VHQVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVKQAPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADESTNTAYMELSSLRSEDTAFYYCARSTYYGGDWYFDVWGQGTT VTVSS 36164 VH STYYGGDWYFDV HCDR3 37 164 VLMGWSCIILFLVATATGVHSDIQLTQSPSSLSASVGDRVTMTCRASSSVSYIHWFQQKPGKAPKPWIYATSNLASGVPVRFSGSGSGTDYTFTISSLQPEDIATYYCQQWTSNPPTF GGGTKLEIK 381.5.3 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSITTAYLQWSSLKASDTAMYYCARHPSYGSGSPNFDYWGQGT LVTVSS 391.5.3 HCDR1 GYSFTSYWIG 40 1.5.3 HCDR2 IIYPGDSDTRYSPSFQG 41 1.5.3 HCDR3HPSYGSGSPNFDY 42 1.5.3 VLDIVMTQTPLSSPVTLGQPASISCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQATQFPLTFGGGTKVEIK 43 1.5.3 LCDR1RSSQSLV YSDGNTYLS 44 1.5.3 LCDR2 KISNRF S 45 1.5.3 LCDR3 VQATQFPLT 46human IgM GCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCGconstant regionTGGCCGTTGGCTGCCTCGCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAA DNAATACAAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGCACGCAGCCACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGCTGAGCTGCCTCCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCTGCGCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTGAGGCAAAGGAGTCTGGGACCACGACCTACAAGGTGACCAGCACACTGACCATCAAAGAGAGCGACTGGCTCAGCCAGAGCATGTTCACCTGCCGCGTGGATCACAGGGGCCTGACCTTCCAGCAGAATGCGTCCTCCATGTGTGGCCCCGATCAAGACACAGCCATCCGGGTCTTCTCCATCCCCCCATCCTTTGCCAGCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGACAGCGTGACCATCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAATGCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAGAGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCAGACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACTTGCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATCACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGACCAGCGCCCCAATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACCGTGTCCGAAGAGGAATGGAACACGGGGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCCTGCCCAACAGGGTCACCGAGAGGACCGTGGACAAGTCCACCGGTAAACCCACCCTGTACAACGTGTCCCTGGTCATGTCCGACACAGC TGGCACCTGCTAC47 human IgN1 GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSconstant regionVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVF AAVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY 48 J-chain DNAATGAAGAACCATTTGCTTTTCTGGGGAGTCCTGGCGGTTTTTATTAAGGCTGTTCATGTGAAAGCCCAAGAAGATGAAAGGATTGTTCTTGTTGACAACAAATGTAAGTGTGCCCGGATTACTTCCAGGATCATCCGTTCTTCCGAAGATCCTAATGAGGACATTGTGGAGAGAAACATCCGAATTATTGTTCCTCTGAACAACAGGGAGAATATCTCTGATCCCACCTCACCATTGAGAACCAGATTTGTGTACCATTTGTCTGACCTCTGTAAAAAATGTGATCCTACAGAAGTGGAGCTGGATAATCAGATAGTTACTGCTACCCAGAGCAATATCTGTGATGAAGACAGTGCTACAGAGACCTGCTACACTTATGACAGAAACAAGTGCTACACAGCTGTGGTCCCACTCGTATATGGTGGTGAGACCAAAATGGTGGAAACAGCCTTAACCCCAGATGCCTGCTATCCTGACTAA 49 J-chain AAmknhllfwgvlavfikavhvkagederivlvdnkckcaritsriirssedpnediverniiivplnnrenisdptsplrtrfvyhlsdlckkcdpteveldnqivtatqsnicdedsatetcytydrnkcytavvplvyggetkmvetaltpdacypd 50 human CD20MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIM amino acidNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIETIPIQEEEEEETETNFPEPPQDQESSP IENDSSP 51RAux-IgM CAGGTTCAGCTGCAGCAGCCCGGAGCCGAGCTGGTCAAACCTGGCGCTAGTGTGAAAAheavy chain TGTCATGCAAGGCATCCGGATACACATTCACTAGCTATAACATGCACTGGGTGAAGCADNA GACCCCCGGCAGGGGTCTGGAGTGGATCGGAGCTATCTACCCCGGCAACGGAGACACATCTTATAATCAGAAGTTTAAAGGCAAGGCCACCCTGACAGCTGATAAGTCCAGCTCTACCGCATACATGCAGCTGAGTTCACTGACAAGCGAGGACTCCGCCGTGTACTATTGCGCCCGGTCCACTTACTATGGCGGAGATTGGTATTTCAATGTGTGGGGAGCAGGCACCACAGTCACCGTCTCGAGCGGCAGTGCTAGCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTCGCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGCCACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGCTGAGCTGCCTCCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCTGCGCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTGAGGCCAAAGAGTCTGGGCCCACGACCTACAAGGTGACCAGCACACTGACCATCAAAGAGAGCGACTGGCTCAGCCAGAGCATGTTCACCTGCCGCGTGGATCACAGGGGCCTGACCTTCCAGCAGAATGCGTCCTCCATGTGTGTCCCCGATCAAGACACAGCCATCCGGGTCTTCGCCATCCCCCCATCCTTTGCCAGCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGACAGCGTGACCATCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAATGCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAGAGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCAGACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACTTGCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATCACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGACCAGCGCCCCAATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACCGTGTCCGAAGAGGAATGGAACACGGGGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCCTGCCCAACAGGGTCACCGAGAGGACCGTGGACAAGTCCACCGGTAAACCCACCCTGTACAACGTGTCCCTGGTCATGTCCGACACAGCTGGCACCTGCTACTGA 52 Ritux-IgMQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTheavy chain AASYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY- 53 Ritux-lightCAAATTGTGCTGTCTCAGAGTCCAGCTATCCTGAGCGCATCTCCCGGAGAGAAGGTGA chain DNACCATGACATGCAGAGCCTCCAGCTCTGTCTCCTACATCCACTGGTTCCAGCAGAAGCCCGGCTCCTCCCCAAAACCCTGGATCTACGCCACCTCTAACCTGGCTAGTGGTGTGCCTGTCAGGTTTAGTGGATCAGGGTCCGGCACCAGCTACTCTCTGACAATCAGCCGGGTGGAGGCTGAAGACGCCGCTACATACTATTGCCAGCAGTGGACTTCTAATCCCCCTACCTTCGGCGGAGGGACAAAGCTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG TTAG 54Ritux-light QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPchain AA VRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC- 55 1.5.3-IgMGAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCCGGCGAGTCCCTGAAGA heavy chainTCTCCTGCAAGGGCTCCGGCTACTCCTTCACCTCCTACTGGATCGGCTGGGTGAGGCA DNAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCATCATCTACCCCGGCGACTCCGACACCAGGTACTCCCCCTCCTTCCAGGGCCAGGTGACCATCTCCGCCGACAAGTCCATCACCACCGCCTACCTGCAGTGGTCCTCCCTGAAGGCCTCCGACACCGCCATGTACTACTGCGCCAGGCACCCCTCCTACGGCTCCGGCTCCCCCAACTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGGCAGTGCTAGCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTCGCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGCCACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGCTGAGCTGCCTCCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCTGCGCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTGAGGCCAAAGAGTCTGGGCCCACGACCTACAAGGTGACCAGCACACTGACCATCAAAGAGAGCGACTGGCTCAGCCAGAGCATGTTCACCTGCCGCGTGGATCACAGGGGCCTGACCTTCCAGCAGAATGCGTCCTCCATGTGTGTCCCCGATCAAGACACAGCCATCCGGGTCTTCGCCATCCCCCCATCCTTTGCCAGCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGACAGCGTGACCATCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAATGCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAGAGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCAGACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACTTGCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATCACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGACCAGCGCCCCAATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACCGTGTCCGAAGAGGAATGGAACACGGGGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCCTGCCCAACAGGGTCACCGAGAGGACCGTGGACAAGTCCACCGGTAAACCCACCCTGTACAACGTGTCCCTGGTCATGTCCGACACAGCTGGCACCTGCTACTGA 56 1.5.3-IgMEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTheavy chain AARYSPSFQGQVTISADKSITTAYLQWSSLKASDTAMYYCARHPSYGSGSPNFDYWGQGTLVTVSSGSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY- 571.5.3 light chainGACATCGTGATGACCCAGACCCCCCTGTCCTCCCCCGTGACCCTGGGCCAGCCCGCCT DNACCATCTCCTGCAGGTCCTCCCAGTCCCTGGTGTACTCCGACGGCAACACCTACCTGTCCTGGCTGCAGCAGAGGCCCGGCCAGCCCCCCAGGCTGCTGATCTACAAGATCTCCAACAGGTTCTCCGGCGTGCCCGACAGGTTCTCCGGCTCCGGCGCCGGCACCGACTTCACCCTGAAGATCTCCAGGGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCGTGCAGGCCACCCAGTTCCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 58 1.5.3 light chainDIVMTQTPLSSPVTLGQPASISCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKISN AARFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCVQATQFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC- 59 human IgA1ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDconstant regionASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPP aa P01876TPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEV DGTCY 60human IgA2 ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDconstant regionASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSL aa P01877HRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY 61 HumanMLLFVLTCLLAVFPAISTKSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQ SecretoryGARGGCITLISSEGYVSSKYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINS ComponentRGLSFDVSLEVSQGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPV PrecursorLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSEEQGGSSRALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGANDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVA AEAQDGPQEA 62human KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSsecretory KYAGRANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLcomponent NDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRmature LDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCRQSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKEDAGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEKVLDSGFREIENKA IQDPR 63precursor MGWSYIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQmodified J- APGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCAchain sequenceRSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVT for V15JITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTP DACYPD 64mature QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTmodified J- HYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVchain sequenceTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKP for V15JGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD 65 PrecursorMKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVER modified J-NIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEchain sequenceDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPDGGGGSGGGGSGGGGS for J15VQVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTF GGGTKLEIK 66mature QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRmodified J- TRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLchain sequenceVYGGETKMVETALTPDACYPDGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSC for J15VKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSAYYDYDGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGGTKLEIK 67 (GGGGS)₃GGGGSGGGGSGGGGS linker 68 J-chain DNAATGGGCTGGTCCTACATCATCCTCTTCCTCGTGGCCACAGCCACAGGCGTCCATAGCC sequence forAGGTGCAGCTGGTGCAGTCCGGCGCCGAAGTGAAGAAGCCTGGCGCCAGCGTGAAGGT V15JGAGCTGCAAGGCTTCCGGCTACACCTTCATCTCCTACACCATGCACTGGGTGAGGCAAGCTCCTGGCCAGGGCCTGGAGTGGATGGGATACATCAACCCTCGGTCCGGCTATACCCACTACAATCAGAAGCTGAAGGACAAGGCCACCCTGACCGCTGACAAGTCCGCCTCCACCGCTTACATGGAGCTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGTGCCAGGTCCGCCTACTACGACTACGACGGATTCGCTTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCCGGAGGAGGAGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGATATCCAGATGACCCAGAGCCCTTCCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACCATCACCTGCAGCGCTTCCTCCTCCGTGTCCTACATGAACTGGTACCAGCAGAAGCCTGGCAAGGCCCCCAAGAGGCTGATCTACGACACCTCCAAGCTGGCCTCCGGAGTGCCTTCCAGGTTCAGCGGCTCCGGCTCCGGAACCGACTTCACCCTGACCATTAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTGGTCCAGCAACCCTCCCACCTTCGGCGGCGGCACAAAGCTGGAGATCAAGGGAGGAGGAGGATCCGGTGGTGGTGGTTCTGGCGGAGGTGGATCCCAAGAAGATGAAAGGATTGTTCTTGTTGACAACAAATGTAAGTGTGCCCGGATTACTTCCAGGATCATCCGTTCTTCCGAAGATCCTAATGAGGACATTGTGGAGAGAAACATCCGAATTATTGTTCCTCTGAACAACAGGGAGAATATCTCTGATCCCACCTCACCATTGAGAACCAGATTTGTGTACCATTTGTCTGACCTCTGTAAAAAATGTGATCCTACAGAAGTGGAGCTGGATAATCAGATAGTTACTGCTACCCAGAGCAATATCTGTGATGAAGACAGTGCTACAGAGACCTGCTACACTTATGACAGAAACAAGTGCTACACAGCTGTGGTCCCACTCGTATATGGTGGTGAGACCAAAATGGTGGAAACAGCCTTAACCCCAGATGCCTGCTATCCTGACTGA 69 J-chain DNAATGAAGAACCATTTGCTTTTCTGGGGAGTCCTGGCGGTTTTTATTAAGGCTGTTCATG sequence forTGAAAGCCCAAGAAGATGAAAGGATTGTTCTTGTTGACAACAAATGTAAGTGTGCCCG J15VGATTACTTCCAGGATCATCCGTTCTTCCGAAGATCCTAATGAGGACATTGTGGAGAGAAACATCCGAATTATTGTTCCTCTGAACAACAGGGAGAATATCTCTGATCCCACCTCACCATTGAGAACCAGATTTGTGTACCATTTGTCTGACCTCTGTAAAAAATGTGATCCTACAGAAGTGGAGCTGGATAATCAGATAGTTACTGCTACCCAGAGCAATATCTGTGATGAAGACAGTGCTACAGAGACCTGCTACACTTATGACAGAAACAAGTGCTACACAGCTGTGGTCCCACTCGTATATGGTGGTGAGACCAAAATGGTGGAAACAGCCTTAACCCCAGATGCCTGCTATCCTGACGGAGGAGGAGGATCCGGTGGTGGTGGTTCTGGCGGAGGTGGATCCCAGGTGCAGCTGGTGCAGTCCGGCGCCGAAGTGAAGAAGCCTGGCGCCAGCGTGAAGGTGAGCTGCAAGGCTTCCGGCTACACCTTCATCTCCTACACCATGCACTGGGTGAGGCAAGCTCCTGGCCAGGGCCTGGAGTGGATGGGATACATCAACCCTCGGTCCGGCTATACCCACTACAATCAGAAGCTGAAGGACAAGGCCACCCTGACCGCTGACAAGTCCGCCTCCACCGCTTACATGGAGCTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGTGCCAGGTCCGCCTACTACGACTACGACGGATTCGCTTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCCGGAGGAGGAGGCAGCGGTGGTGGCGGAAGCGGTGGAGGTGGCAGCGATATCCAGATGACCCAGAGCCCTTCCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACCATCACCTGCAGCGCTTCCTCCTCCGTGTCCTACATGAACTGGTACCAGCAGAAGCCTGGCAAGGCCCCCAAGAGGCTGATCTACGACACCTCCAAGCTGGCCTCCGGAGTGCCTTCCAGGTTCAGCGGCTCCGGCTCCGGAACCGACTTCACCCTGACCATTAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTGGTCCAGCAACCCTCCCACCTTCGGAGGCGGCACAAAGCTGGAGATCAAGTGA

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary embodiments but should be definedonly in accordance with the following claims and their equivalents.

1. A multimeric binding molecule comprising at least two bivalentbinding units, or variants or fragments thereof, wherein each bindingunit comprises at least two heavy chain constant regions or fragmentsthereof, each associated with an antigen-binding domain, wherein atleast one antigen binding domain of the binding molecule is a CD20antigen binding domain comprising six immunoglobulin complementaritydetermining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3,wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 39 orSEQ ID NO: 39 with one or two single amino acid substitutions; the HCDR2comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 40 withone or two single amino acid substitutions; the HCDR3 comprises theamino acid sequence of SEQ ID NO: 41, SEQ ID NO: 41 with one or twosingle amino acid substitutions; the LCDR1 comprises the amino acidsequence of SEQ ID NO: 43, or SEQ ID NO: 43 with one or two single aminoacid substitutions; the LCDR2 comprises the amino acid sequence of SEQID NO: 44 or SEQ ID NO: 44 with one or two single amino acidsubstitutions; and the LCDR3 comprises the amino acid sequence of SEQ IDNO: 45 or SEQ ID NO: 45 with one or two single amino acid substitutions.2. A multimeric binding molecule comprising at least two bivalentbinding units, or variants or fragments thereof, wherein each bindingunit comprises at least two heavy chain constant regions or fragmentsthereof, each associated with an antigen-binding domain, wherein atleast one antigen binding domain of the binding molecule is a CD20antigen binding domain comprising an antibody heavy chain variableregion (VH) and an antibody light chain variable region (VL), whereinthe VH comprises an amino acid sequence at least 80%, at least 85%, atleast 90%, at least 95% or 100% identical to SEQ ID NO: 38, and the VLcomprises an amino acid sequence at least 80%, at least 85%, at least90%, at least 95% or 100% identical to SEQ ID NO:
 42. 3. The bindingmolecule of claim 1, which is a dimeric binding molecule comprising twobivalent IgA binding units or fragments thereof and a J-chain orfragment or variant thereof, wherein each binding unit comprises two IgAheavy chain constant regions or fragments thereof each associated withan antigen-binding domain, wherein the IgA heavy chain constant regionsor fragments thereof each comprise a Cα3-tp domain. 4-5. (canceled) 6.The binding molecule of claim 3, wherein one or more IgA heavy chainconstant regions or fragments thereof further comprise a Cα1 domain, aCα2, or a Cα1 domain and a Cα2 domain. 7-8. (canceled)
 9. The bindingmolecule of claim 3, wherein each binding unit comprises two IgA heavychains each comprising a VH situated amino terminal to the IgA constantregion or fragment thereof, and two immunoglobulin light chains eachcomprising a VL situated amino terminal to an immunoglobulin light chainconstant region.
 10. The binding molecule of claim 1, which is apentameric or a hexameric binding molecule comprising five or sixbivalent IgM binding units, respectively, wherein each binding unitcomprises two IgM heavy chain constant regions or fragments thereof eachassociated with an antigen-binding domain, wherein the IgM heavy chainconstant regions or fragments thereof each comprise a Cα4-tp domain. 11.(canceled)
 12. The binding molecule of claim 10, wherein one or more IgMheavy chain constant regions or fragments thereof further comprise a Cμ3domain, a Cμ2 domain, a Cμ1 domain, or any combination thereof.
 13. Thebinding molecule of claim 10, wherein the binding molecule ispentameric, and further comprises a J-chain, or functional fragmentthereof, or a functional variant thereof.
 14. The binding molecule ofclaim 13, wherein J-chain or fragment thereof comprises the amino acidsequence SEQ ID NO: 49 or a functional fragment thereof.
 15. The bindingmolecule of claim 13, wherein the J-chain or fragment thereof is amodified J-chain further comprising a heterologous polypeptide, whereinthe heterologous polypeptide is directly or indirectly fused via apeptide linker to the J-chain or fragment thereof, wherein the peptidelinker comprises at least 5 amino acids, but no more than 25 aminoacids. 16-17. (canceled)
 18. The binding molecule of claim 15, whereinthe peptide linker consists of GGGGSGGGGSGGGGS (SEQ ID NO: 67).
 19. Thebinding molecule of claim 15, wherein the heterologous polypeptide isfused to the N-terminus of the J-chain or fragment thereof, theC-terminus of the J-chain or fragment thereof, or to both the N-terminusand C-terminus of the J-chain or fragment thereof.
 20. The bindingmolecule of claim 15, wherein the heterologous polypeptide comprises abinding domain.
 21. The binding molecule of claim 20, wherein thebinding domain of the heterologous polypeptide is an antibody orantigen-binding fragment thereof.
 22. The binding molecule of claim 21,wherein the antigen-binding fragment comprises an Fab fragment, an Fab′fragment, an F(ab′)₂ fragment, an Fd fragment, an Fv fragment, asingle-chain Fv (scFv) fragment, a disulfide-linked Fv (sdFv) fragment,or any combination thereof.
 23. The binding molecule of claim 22,wherein the antigen-binding fragment is a scFv fragment.
 24. The bindingmolecule of claim 23, wherein the heterologous polypeptide canspecifically bind to CD3. 25-27. (canceled)
 28. The binding molecule ofclaim 10, wherein the IgM heavy chain constant region is a human IgMconstant region.
 29. The binding molecule of claim 10, wherein eachbinding unit comprises two identical IgM heavy chains each comprising aVH situated amino terminal to the IgM constant region or fragmentthereof, and two identical immunoglobulin light chains each comprising aVL situated amino terminal to an immunoglobulin light chain constantregion.
 30. (canceled)
 31. The binding molecule of claim 29, wherein thetwo IgM heavy chains within at least one binding unit comprise the aminoacid sequence SEQ ID NO: 56, and wherein each light chain comprises theamino acid sequence SEQ ID NO:
 58. 32-35. (canceled)
 36. The bindingmolecule of claim 1, which can direct complement-mediated killing of aCD-20-expressing cell at higher potency than an equivalent amount of amonospecific, bivalent IgG1 antibody comprising a VH having the aminoacid sequence SEQ ID NO: 38 and a VL having the amino acid sequence SEQID NO:
 42. 37. (canceled)
 38. The binding molecule of claim 36, whereinthe CD-20-expressing cell is a lymphoma cell line or a malignant B cellin a subject with cancer. 39-43. (canceled)
 44. The binding molecule ofclaim 38, wherein the cancer is minimally responsive or non-responsiveto rituximab therapy.
 45. The binding molecule of claim 38, wherein thesubject is human.
 46. A composition comprising the binding molecule ofclaim 1 any and a carrier. 47-49. (canceled)
 50. A compositioncomprising (a) a first polynucleotide encoding a heavy chain polypeptidesubunit of the binding molecule of claim 1, wherein the heavy chainpolypeptide subunit comprises an IgM heavy chain constant region orfragment thereof or an IgA heavy chain constant region or fragmentthereof, and at least the antibody VH portion of the CD20 antigenbinding domain; and (b) a second polynucleotide encoding a light chainpolypeptide subunit, wherein the light chain polypeptide subunitcomprises the antibody VL portion of the CD20 antigen binding domain.51-68. (canceled)
 69. A host cell comprising the composition of claim50, wherein the host cell can express the binding molecule.
 70. A methodof producing the binding molecule comprising culturing the host cell ofclaim 69, and recovering the binding molecule.
 71. A method fordirecting complement-mediated, T-cell-mediated, or bothcomplement-mediated and T-cell-mediated killing of a CD20-expressingcell comprising contacting a CD20-expressing cell with the bindingmolecule of claim 1, wherein the binding molecule can directcomplement-mediated killing of a CD-20-expressing cell at higher potencythan an equivalent amount of a monospecific, bivalent IgG1 antibody orfragment thereof that specifically binds to the same CD20 epitope as theCD20 antigen binding domain. 72-79. (canceled)
 80. A method fordirecting complement-mediated, T-cell-mediated, or bothcomplement-mediated and T-cell-mediated killing of a CD20-expressingcell comprising contacting a CD20-expressing cell with a dimeric,pentameric, or hexameric binding molecule comprising two, five, or sixbivalent binding units, respectively, wherein each binding unitcomprises two IgA or IgM heavy chain constant regions or fragmentsthereof and two antigen binding domains, wherein at least one antigenbinding domain of the binding molecule is a CD20 antigen binding domain,and wherein the binding molecule can direct complement-mediated killingof a CD-20-expressing cell at higher potency than an equivalent amountof a monospecific, bivalent IgG1 antibody or fragment thereof thatspecifically binds to the same CD20 epitope as the CD20 antigen bindingdomain. 81-121. (canceled)